Compositions and Methods for Treating Atherosclerosis

ABSTRACT

Peptides and mimetics of selected domains of mammalian serum amyloid A isoform 2.1 (SAA2.1) and compounds and compositions thereof are provided that enhance the effect on macrophage cholesterol ester hydrolase activity and/or inhibit acyl CoA:cholesterol acyl transferase activity. Methods of using these compositions in the treatment and/or prevention of atherosclerosis as well as coronary heart disease and cardiovascular disease are also provided.

INTRODUCTION

This patent application is a continuation of U.S. application Ser. No.11/872,309 filed Oct. 15, 2007, which is a divisional of U.S.application Ser. No. 10/866,330, filed Jun. 10, 2004, now issued as U.S.Pat. No. 7,291,590, which claims the benefit of priority from U.S.Provisional patent application Ser. No. 60/544,565, filed Feb. 13, 2004and U.S. Provisional patent application Ser. No. 60/478,131, filed Jun.12, 2003, each of which is herein incorporated by reference in itsentirety.

FIELD OF THE INVENTION

Peptides useful in inhibiting the storage of cholesterol andpotentiating the mobilization and release of cholesterol frominflammatory or atherosclerotic sites in a subject have been identified.The present invention relates to isolated peptides, more preferablysynthetic peptides including chemically and recombinantly synthesizedpeptides, compounds, and mimetics of these peptides and compounds, andpharmaceutical compositions comprising one or more of these peptides ora portion thereof, or compounds, or mimetics of these, and methods ofusing these peptides or mimetics thereof, compounds or mimetics thereofand pharmaceutical compositions in the treatment and/or prevention ofatherosclerosis and inflammation, as well as coronary heart disease andcardiovascular disease.

BACKGROUND OF THE INVENTION

Cardiovascular disease, including coronary heart disease caused byatherosclerosis, is the single largest killer of adults in North America(2002 Heart and Stroke Statistical Update). The development andprogression of atherosclerosis in coronary arteries can lead to heartattacks and angina. In 1999 it was estimated that 12.6 million Americanshad coronary heart disease. Approximately 1 in 5 deaths in 1999 were dueto coronary heart disease, with a total US and Canadian mortality ofover 500,000 and 42,000 individuals, respectively. It is estimated thatover 102 million American adults have blood cholesterol levels that areeither border-line high risk, or high risk of developing coronary heartdisease. In addition to the immediate social and economic burden thatheart attacks have on our health care system, there also is theconsiderable cost associated with the aftermath of a coronary heartdisease event. About 25% of males and 38% of females will die one yearafter a heart attack, and death by coronary heart disease tends to occurduring a person's peak productive years (BRFSS [1997], MMWR vol. 49, No.SS-2, Mar. 24, 2000, CDC/NCHS). There is also a further economic burdenof coronary heart disease associated with premature and permanentdisability of the labor force. In 1998, over $10 billion was paid toMedicare beneficiaries for coronary heart disease (Health Care FinancingReview, Statistical Supplement [2000], HFCA).

Patients currently have a choice of a number of different drugs to treatcardiovascular disease/coronary heart disease. These drugs fall intovarious classes, including antihypertensives and antihyperlipidemics.Although these products have been shown to be beneficial in reducing theprogression of coronary heart disease and preventing heart attacks, theycan be limited in their effectiveness in some individuals because of lowtolerability and, in some cases, mitigation of drug efficacy by thecompensatory effects of the liver (Turley, S. D. (2002) Am. J. ManagedCare 8 (2 Suppl):S29-32).

The accumulation of lipids, especially cholesterol, in several aorticand arterial cell-types, such as macrophages and smooth muscle cells, isthe defining pathologic feature of atherosclerosis (Gotlieb et al.(1999) Blood Vessels. In Pathology. Rubin, E. and Farber, J. L.,editors. Lippincott-Raven, Philadelphia, New York. 481-530). Majorinvestigative efforts are being expended to understand two centralissues related to this problem. The first relates to the mechanism bywhich cholesterol is delivered to, and taken up by, these cells. Thesecond relates to the process by which these cells export and ridthemselves of excessive cholesterol. In the treatment and prevention ofatherosclerosis, one of the aims is to limit the intracellularaccumulation of large quantities of cholesterol that adversely influencethe viability of these cells, thereby eventually altering the structuralintegrity of the blood vessels.

An analogous set of events occurs in acute tissue injury. Such injuriesresult in local cell death and set in motion local inflammation and thesystemic acute phase response (Fantone, J. C. and Ward, P. A. (1994)Inflammation. In Pathology. Rubin, E. and Farber, J. editors.Lippincott, Philadelphia. 32-6). Alterations in local cholesterolprocessing are important components of this process. At sites of acutetissue injury, dying cells release large quantities of cell debris thatincludes cell membrane fragments rich in cholesterol (Fantone, J. C. andWard, P. A. (1994) Inflammation. In Pathology. Rubin, E. and Farber, J.editors. Lippincott, Philadelphia. 32-6). As part of acute inflammation,macrophages arriving at sites of injury ingest these fragments forfurther processing and thereby acquire a considerable cholesterol load,becoming foam cells, analogous to those seen in atherosclerosis. Duringacute tissue injury and the consequent acute inflammatory process, acholesterol removal mechanism is required to mobilize the cholesteroleither for excretion or re-use.

The physiological role of one of the major acute phase (AP) proteinssynthesized by the liver in response to tissue injury, serum amyloid A(SAA), is directly related to these events and processes. SAA representsa group of four polymorphic proteins, encoded by a multigene family,that have been conserved for over 600 million years (Jensen et al.(1997) J. Immunol. 158:384-392; Santiago et al. (2000) J. Exp. Zool.288: 3335-344). Isoforms SAA1.1 and SAA2.1 are present in plasma inacute phase tissue injury and are the most thoroughly investigated.

The nomenclature for serum amyloid A was revised in 1999, as there was arecognized need by researchers for a systematic nomenclature of themultiple SAA genes in human and animal models and for their allelicvariants (Amyloid: Int. J. Exp. Clin. Invest. 1999 6:67-70). The majorrevision was the re-designation of the mouse Saa1 and Saa2 genes. Basedupon chromosomal mapping, it appears that the mouse Saa2 locuscorresponds to human SAA1. Therefore, the mouse nomenclature was changedto be fully compatible with the human nomenclature.

The following Tables set forth the revised nomenclature for SAA mouseand human proteins as well as their corresponding sequences. Thesetables are based upon the disclosure in 1999 in Amyloid: Int. J. Exp.Clin. Invest. 6:67-70. The tables presented herein have been modified,however, to clarify alignment and provide numbering for residue (−1) ofmouse isoform SAA3 comprising an additional amino acid.

TABLE I Mouse SAA proteins New Old −1 1 2 3 4 5 6 7 8 9 10 11 12 SAA1.1SAA2 G F F S F I G E A F Q G SAA1.2 SJL/J SAA1.3 mc1 S SAA1.4 mc2 SSAA1.5 mm1 V H SAA1.6 mm2 SAA2.1 SAA1 V H SAA2.2 CE/J V H L SAA3 Q R W VQ M K G SAA4 SAA5 D W Y F R 13 14 15 16 17 18 19 20 21 22 23 24 25SAA1.1 SAA2 A G D M W R A Y T D M K E SAA1.2 SJL/J SAA1.3 mc1 R SAA1.4mc2 SAA1.5 mm1 SAA1.6 mm2 SAA2.1 SAA1 SAA2.2 CE/J SAA3 S R S K SAA4 SAA5T W L R N L 26 27 28 29 30 31 32 33 34 35 36 37 38 SAA1.1 SAA2 A G W K DG D K Y F H A R SAA1.2 SJL/J SAA1.3 mc1 R SAA1.4 mc2 SAA1.5 mm1 SAA1.6mm2 SAA2.1 SAA1 N N S SAA2.2 CE/J SAA3 S SAA4 SAA5 N Y Q N A Q Y 39 4041 42 43 44 45 46 47 48 49 50 51 SAA1.1 SAA2 G N Y D A A Q R G P G G VSAA1.2 SJL/J SAA1.3 mc1 SAA1.4 mc2 SAA1.5 mm1 SAA1.6 mm2 SAA2.1 SAA1SAA2.2 CE/J SAA3 R A SAA4 SAA5 E Q S I 52 53 54 55 56 57 58 59 60 61 6263 64 SAA1.1 SAA2 W A A E K I S D A R E S F SAA1.2 SJL/J SAA1.3 mc1SAA1.4 mc2 G SAA1.5 mm1 SAA1.6 mm2 SAA2.1 SAA1 G A SAA2.2 CE/J A SAA3 KV V SAA4 SAA5 K I T S K Y 65 66 67 68 69 70 71 72 73 74 75 76 77 SAA1.1SAA2 Q E F F G R G H E D T M A SAA1.2 SJL/J SAA1.3 mc1 SAA1.4 mc2 SAA1.5mm1 SAA1.6 mm2 SAA2.1 SAA1 I SAA2.2 CE/J SAA3 K T H A S R SAA4 SAA5 G LL N H L T L Q ↑ [NRYYFGIR] 78 79 80 81 82 83 84 85 86 87 88 89 90 SAA1.1SAA2 D Q E A N R H G R S G K D SAA1.2 SJL/J SAA1.3 mc1 SAA1.4 mc2 SAA1.5mm1 SAA1.6 mm2 SAA2.1 SAA1 SAA2.2 CE/J SAA3 F E W SAA4 SAA5 T K E E W N91 92 93 94 95 96 97 98 99 100 101 102 103 SAA1.1 SAA2 P N Y Y R P P G LP A K Y SAA1.2 SJL/J D SAA1.3 mc1 SAA1.4 mc2 SAA1.5 mm1 SAA1.6 mm2 DSAA2.1 SAA1 D SAA2.2 CE/J D SAA3 H F A K R SAA4 SAA5 H F E E F

TABLE II Human SAA proteins New Old 1 2 3 4 5 6 7 8 9 10 11 12 13 14 1516 SAA1.1 SAA1α R S F F S F L G E A F D G A R D SAA1.2 SAA1β SAA1.3SAA1γ SAA1.4 SAA1δ SAA1.5 SAA1β SAA2.1 SAA2α SAA2.2 SAA2β SAA4 E S W R SF F K E A L Q G V G D 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32SAA1.1 SAA1α M W R A Y S D M R E A N Y I G S SAA1.2 SAA1β SAA1.3 SAA1γSAA1.4 SAA1δ SAA1.5 SAA1β SAA2.1 SAA2α SAA2.2 SAA2β SAA4 M G R A Y M D IM I S M H Q N S 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 New OldD K Y F H A R G N Y D A A K R G SAA1.1 SAA1α SAA1.2 SAA1β SAA1.3 SAA1γSAA1.4 SAA1δ SAA1.5 SAA1β SAA2.1 SAA2α SAA2.2 SAA2β SAA4 N R Y L Y A R GN Y D A A Q R G 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 SAA1.1SAA1α P G G V W A A E A I S D A R E N SAA1.2 SAA1β A V SAA1.3 SAA1γ ASAA1.4 SAA1δ A V N SAA1.5 SAA1β A V SAA2.1 SAA2α A V N SAA2.2 SAA2β A VN SAA4 P G G V W A A K L I S R S R V Y 65 66 67 68 69 70 71 72 73 74 7576 77 78 79 80 SAA1.1 SAA1α I Q R F F G H G A E D S L A D Q SAA1.2 SAA1βD SAA1.3 SAA1γ SAA1.4 SAA1δ SAA1.5 SAA1β SAA2.1 SAA2α L T SAA2.2 SAA2β LT R SAA4 L Q G L I S T V L E D S K S N E ↑ [DYYLEFGNS] 81 82 83 84 85 8687 88 89 90 91 92 93 94 95 96 SAA1.1 SAA1α A A N E W G R S G K D P N H FR SAA1.2 SAA1β SAA1.3 SAA1γ SAA1.4 SAA1δ SAA1.5 SAA1β SAA2.1 SAA2α K RSAA2.2 SAA2β K R SAA4 K A E E W G R S G K D P D R F R 97 98 99 100 101102 103 104 SAA1.1 SAA1α P A G L P E K Y SAA1.2 SAA1β SAA1.3 SAA1γSAA1.4 SAA1δ SAA1.5 SAA1β SAA2.1 SAA2α SAA2.2 SAA2β SAA4 P D G L P K K YThe nomenclature for SAA proteins employed in this patent applicationcorresponds to the revised nomenclature as set forth in the aboveTables. However, it must be appreciated that journal referencespublished prior to this 1999 revision and patent applications filedprior to this 1999 revision may use the old nomenclature, thus, forexample referring to mouse Saa1 as mouse Saa2 and vice versa.

SAA isoforms SAA1.1 and SAA2.1 are produced primarily by hepatocytes inresponse to various causes of tissue injury and inflammation (Morrow etal. (1981) Proc. Natl. Acad. Sci. USA 78:4718-4722). Synthesis of SAA1.1and 2.1 by the liver is induced by cytokines such as interleukin-1,interleukin-6, and tumor necrosis factor, which are released byactivated macrophages, and which act through a set of downstreameffectors in the hepatocyte cytoplasm and nucleus (Edbrooke et al.(1991) Cytokine 3:380-388; Betts et al. (1993) J. Biol. Chem.268:25624-25631; Ray et al. (1999) J. Biol. Chem. 274:4300-430810; andSipe et al. (1987) Lymphokine Res. 6:93-101). Maximum transcriptionrates for the SAA1.1 and 2.1 genes are seen 3-4 hours following tissueinjury, and within 18-24 hours of injury the plasma concentration ofthese two proteins rises from 1-5 μg/mL to 500-1000 μg/mL (500-1000-foldincrease) (McAdam et al. (1978) J. Clin. Invest. 61:390-394; McAdam, K.P., Sipe, J. D. (1976) J. Exp. Med. 144:1121-1127). Once secreted fromhepatocytes, SAA1.1 and 2.1 are found predominantly in the high densitylipoprotein (HDL) fraction and form 30-80% of the HDL apolipoproteins,resulting in a major reorganization of the apolipoprotein composition ofthe HDL fraction (Benditt et al. (1979) Proc. Natl. Acad. Sci. USA 76:4092-4096; Hoffman, J. S. and Benditt, E. P. (1982) J. Biol. Chem.257:10518-10522).

At present there is debate whether the observed increase in SAAexpression during tissue injury is associated with a beneficial roleagainst atherosclerotic lesions, or whether increased SAA levels are infact associated with a role in developing atherosclerosis.

Elevated levels of SAA isoforms are observed during the earlypathological vascular events leading to atherosclerosis before clinicalsymptoms are evident. (reviewed in Kisilevsky, R. and Tam, S.-P. (2002)Pediatric Pathol. and Mol. Med. 21: 291-303). This elevation has ledsome researchers to suggest that SAA levels may play a causative,contributing role in atherogenesis (Jousilahti et al. (2001)Atherosclerosis 156:451-456; Kumon et al. (1998) Scand. J. Immunol.48:419-424; Liuzzo et al. (1994) N. Engl. J. Med. 331:417-424; Ridker etal. (1998) Circulation 98:839-844; Rosenthal, C. J. and Franklin, E. C.(1975) J. Clin. Invest. 55:746-753; Steinmetz et al. (1989) BiochimBiophys. Acta. 1006:173-178; Van Lenten et al. (1995) J. Clin. Invest.96:2758-2767).

However, there have also been reports of SAA and isoforms thereofpromoting the efflux of cholesterol from macrophages.

For example, high density lipoprotein-serum amyloid A (HDL-SAA) has beenshown to have reduced ability to accept cholesterol from low densitylipoprotein/very low density lipoprotein (LDL/VLDL), ensuring that HDL,in its afferent route, arrives at macrophages carrying as littlecholesterol as possible (Kisilevsky et al. (1996) Amyloid 3: 252-260).Thus, this form of HDL has a greater capacity to accept cholesterol fromcholesterol-laden macrophages. HDL-SAA has also been demonstrated tohave a 3 to 4-fold higher affinity for macrophages when compared to HDLalone. Further, an increase was observed in the number of HDL-SAAbinding sites on macrophages obtained from animals with an APinflammatory reaction. Competition studies with macrophages (Kisilevsky,R. and Subrahmanyan, L. (1992) Lab. Invest. 66: 778-785) showed thatunlabelled HDL-SAA, but not HDL alone, effectively displacedradiolabeled HDL-SAA. This preferential displacement by HDL-SAA islikely indicative of the presence of SAA receptors on the macrophages.Such SAA receptors are separate and additional to the binding sites forapoA-1 on the macrophages (Kisilevsky, R. and Subrahmanyan, L. (1992)Lab. Invest. 66: 778-785; U.S. Pat. No. 6,004,936). The presence of SAAreceptors is further supported by the demonstration that HDL-SAA was inclathrin coated pits shortly after binding to macrophages. These pitsand the resulting endosomes are consistent with the concept ofreceptor-mediated endocytosis, a process that is dependent on cellsurface heparin sulphate, to which SAA binds effectively (Ancsin, J. andKisilevsky, R. (1999) J. Biol. Chem. 274:7172-7181; Rocken, C. andKisilevsky, R. (1997) Amyloid 4: 259-273).

More recent studies have demonstrated that SAA enhances HDL uptake bymacrophages (Banka et al. (1995) J. Lipid Res. 36:1058-10865) and has anaffinity for cholesterol (Liang, J. S. and Sipe, J. D. (1995) J. LipidRes. 36:37-46). Using synthetic peptides corresponding to residues 1-18and 40-63 of human apoSAA₁ (now referred to as SAA1.1) and residues 1-18of human apoSAA₄ (now referred to as SAA4) it was shown that apoSAA₁ butnot apoSAA₄ binds cholesterol at the amino terminal region (Liang et al.(1996) J. Lipid Res. 37:2109-2116).

Furthermore murine SAA2.1, but not murine SAA1.1, was shown to inhibitmacrophage acyl CoA:cholesterol acyl transferase (ACAT) activity inculture in intact murine macrophages and in their post-nuclearhomogenates in a dose-dependent manner (Ely et al. (2001) Amyloid8:169-181). Further examination of cyanogen bromide generated cleavagefragments of murine SAA2.1 purified by reverse phase HPLC showed murineSAA2.1₁₋₁₆ to have a profound effect inhibiting ACAT activity in adose-dependent manner. In contrast, murine SAA2.1₂₄₋₁₀₃ exhibited noinhibitory effect on ACAT activity (Ely et al. (2001) Amyloid8:169-181).

Murine SAA2.1 has also been shown to stimulate hepatic, macrophage, andpancreatic cholesterol esterase activities in vitro (Lindhorst et al.(1997) Biochim. Biophys. Acta 1339:143-154; Ely et al. (2001) Amyloid8:169-181; Tam et al. (2002) J. Lipid Res. 43:1410-1420). This effectwas shown to reside in the 80 residue COOH-terminal region of murineSAA2.1 liberated by cyanogen bromide cleavage (Ely et al. (2001) Amyloid8:169-181). This 80 residue region comprises residues 24-103 of murineSAA2.1.

The ability of HDL-SAA and liposomes containing murine SAA2.1 to cause amarked reduction of acyl CoA:cholesterol acyl transferase activity andenhancement of cholesterol efflux activity was confirmed in macrophagesin culture (Tam et al. (2002) J. Lipid Res. 43:1410-1420). Intravenousinjection of [³H]-cholesterol-loaded macrophages into inflamed mice hasalso been reported to result in a 3- to 3.5-fold increase in the amountof radiolabeled cholesterol released into the plasma when compared tosimilarly treated un-inflamed control animals (Tam et al. J. Lipid Res.2002 43:1410-1420). In this study, macrophage cholesterol efflux wasshown to be coupled to the ATP-binding cassette transporter, ABCA1,which is an important protein for the initial step of the reversecholesterol transport pathway. Furthermore, [³H]-cholesterol-ladenmacrophages, when pre-treated with HDL-SAA2.1 (murine) in tissue cultureand then injected into un-inflamed mice, rapidly released theircholesterol into the plasma (Tam et al. (2002) J. Lipid Res.43:1410-1420). This result was not observed when macrophages weretreated with HDL alone.

Thus, isoforms SAA1.1 and SAA2.1 are up-regulated during inflammation;they are evolutionarily conserved; and they are predominantly associatedwith HDL and HDL's established role in the reverse cholesterol transportpathway (Lindhorst et al. (1997) Biochim. Biophys. Acta 1339:143-154;Kisilevsky, R. (1991) Med. Hypotheses 35: 337-341; Kisilevsky, R. et al.(1996) Amyloid 3: 252-260; and Kisilevsky, R. and Subrahmanyan, L.(1992) Lab. Invest. 66: 778-785).

U.S. Pat. No. 5,318,958 discloses methods of potentiating the releaseand collection of macrophage cholesterol in vivo by administering aneffective amount of HDL bound to a ligand having serum amyloid Aaffinity for HDL. A preferred ligand of this method taught in thispatent is serum amyloid A itself.

U.S. Pat. No. 6,004,936 describes similar methods to U.S. Pat. No.5,318,958. However, in the method claimed in U.S. Pat. No. 6,004,936,the ligand having serum amyloid affinity is not bound to HDL prior toadministration. This patent teaches that preferred ligands having serumamyloid affinity are non-amyloidogenic isoforms of serum amyloid A suchas SAA2.1.

SUMMARY OF THE INVENTION

Selected peptide domains of mammalian serum amyloid A isoforms 2.1(SAA2.1) and 1.1 (SAA1.1) and mimetics thereof are demonstrated hereinto have a potent enhancing effect on macrophage cholesterol esterhydrolase activity (CEH) and/or an inhibiting effect on acylCoA:cholesterol acyl transferase (ACAT) activity. As shown herein, thesepeptides and mimetics thereof shift macrophage cholesterol into atransportable form that is then rapidly exported from the cell in thepresence of a cholesterol transporter and a cholesterol acceptor, highdensity lipoprotein (HDL). Thus, these peptides and mimetics thereof areuseful in methods of inhibiting the storage of cholesterol andpotentiating the mobilization and release of cholesterol frominflammatory or atherosclerotic sites in a subject.

Accordingly, the present invention provides peptides and compounds andmimetics of these peptides and compounds and pharmaceutical compositionscomprising these peptides or portions thereof, compounds and mimetics ofthese peptides or portions thereof or compounds, and methods for use ofthese peptides, compounds and pharmaceutical compositions to modify theactivity of the macrophage cholesterol metabolizing enzyme cholesterolester hydrolase and/or acyl CoA:cholesterol acyl transferase.

One aspect of the present invention relates to a peptide, a peptidevariant or mimetic thereof of the cholesterol ester hydrolase enhancingdomain or the acyl CoA:cholesterol acyl transferase inhibitory domain ofSAA proteins. Cholesterol ester hydrolase enhancing domains have nowbeen identified as residing in residues 74-103 of the C-terminus ofmurine SAA2.1 and residues 77-103 of the C-terminus of murine SAA1.1. Anacyl CoA:cholesterol acyl transferase inhibitory domain resides inresidues 1-16 of the N-terminus of murine SAA2.1 and in residues 1-23 ofthe N-terminus of human SAA1.1 and SAA2.1. Preferred peptides ormimetics thereof capable of enhancing cholesterol ester hydrolaseactivity include an isolated peptide or a mimetic thereof comprising aformula X₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁X₁₂X₁₃X₁₄X₁₅X₁₆X₁₇X₁₈ (SEQ ID NO:29) ora portion thereof wherein X₁ and X₉, X₁₂ or X₁₈ are amino acids capableof forming a salt bridge, X₆ is glutamic acid or lysine or an amino acidwhich is a conservative substitution thereof, and X₂, X₃, X₄, X₅, X₇,X₈, X₁₀, X₁₁, X₁₃, X₁₄, X₁₅, X₁₆, and X₁₇ are independently any aminoacid. Also preferred are peptides comprisingDTIADQEANRHGRSGKDPNYYRPPGLPDKY (SEQ ID NO:4);ADQEANRHGRSGKDPNYYRPPGLPDKY (SEQ ID NO:8); ADQEANRHGRSGKDPNYYRPPGLPAKY(D-form; SEQ ID NO:10); ADQEANRHGRSGKDPNYYR (SEQ ID NO:25);ADQAANKWGRSGRDPNHFR (SEQ ID NO:11); ADQAANEWGRSGKDPNHFR (SEQ ID NO:12);or DQAANKWGRSGRDPNHFR (SEQ ID NO:26), or mimetics thereof and peptidevariant or mimetics thereof of a peptide comprisingDTIADQEANRHGRSGKDPNYYRPPGLPDKY (SEQ ID NO:4);ADQEANRHGRSGKDPNYYRPPGLPDKY (SEQ ID NO:8); ADQEANRHGRSGKDPNYYRPPGLPAKY(SEQ ID NO:9); ADQEANRHGRSGKDPNYYRPPGLPAKY (D-form; SEQ ID NO:10);ADQAANEWGRSGKDPNHFRPAGLPEKY (SEQ ID NO:24); ADQEANRHGRSGKDPNYYR (SEQ IDNO:25); ADQAANKWGRSGRDPNHFR (SEQ ID NO:11); ADQAANEWGRSGKDPNHFR (SEQ IDNO:12); or DQAANKWGRSGRDPNHFR (SEQ ID NO:26) or a portion thereof.Excluded from the scope of peptides of the present invention capable ofenhancing cholesterol ester hydrolase activity are those isolatedpeptides consisting ofGFFSFIGEAFQGAGDMWRAYTDMKEAGWKDGDKYFHARGNYDAAQRGPGGVWAAEKISDARESFQEFFGRGHEDTMADQEANRHGRSGKDPNYYRPPGLPAKY (full length murine SAA1.1;SEQ ID NO:18);GFFSFVHEAFQGAGDMWRAYTDMKEANWKNSDKYFHARGNYDAAQRGPGGVWAAEKISDGREAFQEFFGRGHEDTIADQEANRHGRSGKDPNYYRPPGLPDKY (full length murine SAA2.1;SEQ ID NO:19);RSFFSFLGEAFDGARDMWRAYSDMREANYIGSDKYFHARGNYDAAKRGPGGVWAAEAISDARENIQRFFGHGAEDSLADQAANEWGRSGKDPNHFRPAGLPEKY (full length human SAA1.1;SEQ ID NO:20);RSFFSFLGEAFDGARDMWRAYSDMREANYIGSDKYFHARGNYDAAKRGPGGAWAAEVISNARENIQRLTGHGAEDSLADQAANKWGRSGRDPNHFRPAGLPEKY (full length human SAA2.1;SEQ ID NO:21);KEAGWKDGDKYFHARGNYDAAQRGPGGVWAAEKISDARESFQEFFGRGHEDTMADQEANRHGRSGKDPNYYRPPGLPAKY (SEQ ID NO:22);KEANWKNSDKYFHARGNYDAAQRGPGGVWAAEKISDGREAFQEFFGRGHEDTIADQEANRHGRSGKDPNYYRPPGLPDKY (SEQ ID NO:23); ADQEANRHGRSGKDPNYYRPPGLPAKY (SEQID NO:9); or ADQAANEWGRSGKDPNHFRPAGLPEKY (SEQ ID NO:24).

Preferred peptides of the present invention capable of inhibiting acylCoA:cholesterol acyl transferase activity include an isolated peptide ora peptide variant or portion thereof, or a similar region of theN-terminus of human SAA1.1 or SAA2.1 or peptide variants or portionsthereof comprising a formula (X)_(n)FFX₁FX₂X₃X₄X₅FX₆ or a portionthereof wherein F is phenylalanine or an amino acid which is aconservative substitution thereof, and n is 1 or 2. When n is 1, theisolated peptide comprises XFFX₁FX₂X₃X₄X₅FX₆ (SEQ ID NO:13) wherein F isphenylalanine or an amino acid which is a conservative substitutionthereof, X, X₁, X₄, X₅ and X₆ are independently any amino acid, X₂ is ahydrophobic or nonpolar amino acid, and X₃ is histidine or an amino acidwhich is a conservative substitution thereof. When n is 2, the isolatedpeptide comprises X_(a)X_(b)FFX₁FX₂X₃X₄X₅FX₆ (SEQ ID NO:14), wherein Fis phenylalanine or an amino acid which is a conservative substitutionthereof, X_(a) and X₆ are amino acids capable of forming a salt bridge,and X_(b), X, X₁, X₂, X₃, X₄ and X₅ are independently any amino acid.Also preferred are isolated peptides comprising GFFSFVHEAFQGAGDMWRAY(SEQ ID NO:1), RSFFSFLGEAFDGARDMWRAYSD (SEQ ID NO:6), orRGFFSFIGEAFQGAGDMWRAY (SEQ ID NO:7) or a peptide variant of one of thesepeptides or a portion thereof. Excluded from the scope of the peptidesof the present invention capable of inhibiting acyl CoA:cholesterol acyltransferase activity are those isolated peptides consisting ofGFFSFVHEAFQGAGDM (SEQ ID NO:15), GFFSFIGEAFQGAGDM (SEQ ID NO:16),RSFFSFLGEAFDGARDMW (SEQ ID NO:17),GFFSFIGEAFQGAGDMWRAYTDMKEAGWKDGDKYFHARGNYDAAQRGPGGVWAAEKISDARESFQEFFGRGHEDTMADQEANRHGRSGKDPNYYRPPGLPAKY (full length murine SAA1.1;SEQ ID NO:18);GFFSFVHEAFQGAGDMWRAYTDMKEANWKNSDKYFHARGNYDAAQRGPGGVWAAEKISDGREAFQEFFGRGHEDTIADQEANRHGRSGKDPNYYRPPGLPDKY (full length murine SAA2.1;SEQ ID NO:19);RSFFSFLGEAFDGARDMWRAYSDMREANYIGSDKYFHARGNYDAAKRGPGGVWAAEAISDARENIQRFFGHGAEDSLADQAANEWGRSGKDPNHFRPAGLPEKY (full length human SAA1.1;SEQ ID NO:20); orRSFFSFLGEAFDGARDMWRAYSDMREANYIGSDKYFHARGNYDAAKRGPGGAWAAEVISNARENIQRLTGHGAEDSLADQAANKWGRSGRDPNHFRPAGLPEKY (full length human SAA2.1;SEQ ID NO:21).

Preferred variants include, but are not limited to, peptides comprisingone or more D amino acids, which are equally effective but lesssusceptible to degradation in vivo, and cyclic peptides.

Also preferred is a variant comprising two or more linked or conjugatedpeptides of the present invention. Particularly preferred is a variantcomprising a peptide capable of enhancing cholesterol ester hydrolaseactivity linked or conjugated to a peptide capable of inhibiting acylCoA:cholesterol acyl transferase activity.

The present invention also relates to mimetics of any of the abovepeptides, peptide variants or portions thereof.

Another aspect of the present invention relates to compounds with aformula of Y—Z or Q-Y—Z, wherein Y comprises an isolated peptide ormimetic of the present invention with cholesterol ester hydrolaseenhancing activity and/or acyl CoA:cholesterol acyl transferaseinhibitory activity; Z comprises a compound linked to Y that enhancesthe performance of Y; and in embodiments comprising Q, Q may compriseanother compound linked to Y—Z which also enhances performance of theQ-Y—Z compound. Q may be identical to Z or different from Z. Exemplary Zor Q compounds include, but are not limited to a targeting agent, asecond agent for treatment of atherosclerosis, cardiovascular disease orcoronary heart disease, an agent which enhances solubility, absorption,distribution, half-life, bioavailability, stability, activity and/orefficacy, or an agent which reduces toxicity or side effects of thecompound. Exemplary targeting agents of Z and/or Q include macrophagetargeting agents such as, for example, a liposome, a microsphere, or aligand for a SAA receptor, hepatic targeting agents, antibodies andactive fragments thereof such as, for example, Fab fragments, andadditional agents specific to atherosclerotic plaques and/orinflammatory sites.

Another aspect of the present invention relates to pharmaceuticalcompositions comprising a peptide, peptide variant or portion thereof, aY—Z or Q-Y—Z compound, or a mimetic of these, which inhibits acylCoA:cholesterol acyl transferase activity and/or enhances cholesterolester hydrolase activity. Pharmaceutical compositions of the presentinvention further comprise a vehicle suitable pharmaceutically for invivo administration. In one embodiment, the isolated peptide or mimeticthereof or the compound is complexed with a lipid. A phospholipidvesicle which encapsulates the peptide or mimetic thereof or thecompound can also be used.

Another aspect of the present invention relates to the use of thesepeptides, compounds and mimetics of these, or pharmaceuticalcompositions comprising these peptides, compounds and mimetics of these,to modify an activity of a cholesterol-metabolizing enzyme. Inparticular, the activity of cholesterol ester hydrolase and/or acylCoA:cholesterol acyl transferase can be modified using a peptide,compound or mimetic of these, or a pharmaceutical composition comprisinga peptide, compound or mimetic of these of the present invention. In apreferred embodiment of the present invention, the enzymatic activity ismodified in vivo. More preferred is modification of the enzymaticactivity in humans.

Another aspect of the present invention relates to use of thesepeptides, compounds and mimetics of these, or pharmaceuticalcompositions comprising these peptides, compounds and mimetics of these,to increase and/or promote the mobilization and efflux of storedcholesterol from macrophages located in atherosclerotic plaques. In apreferred embodiment of the present invention, the increase and/orpromotion of the mobilization and efflux of stored cholesterol frommacrophages located in atherosclerotic plaques occurs in vivo. Morepreferred is increase and/or promotion of the mobilization and efflux ofstored cholesterol from macrophages located in atherosclerotic plaquesin humans.

Another aspect of the present invention relates to use of thesepeptides, compounds and mimetics of these or pharmaceutical compositionscomprising these peptides, compounds and mimetics of these to increaseand/or promote the mobilization and efflux of stored cholesterol frommacrophages located at sites of inflammation. In a preferred embodimentof the present invention, the increase and/or promotion of themobilization and efflux of stored cholesterol from macrophages locatedat sites of inflammation occurs in vivo. More preferred is increaseand/or promotion of the mobilization and efflux of stored cholesterolfrom macrophages located at sites of inflammation in humans.

Another aspect of the present invention relates to methods for treatingor preventing atherosclerosis in a subject comprising administering tothe subject a peptide, compound, or a mimetic of these or apharmaceutical composition of the present invention. In a preferredembodiment the subject is a human.

Another aspect of the present invention relates to methods for treatmentof cardiovascular disease comprising administering to a subject apeptide, compound, or mimetic of these or a pharmaceutical compositionof the present invention. In a preferred embodiment the subject is ahuman.

Another aspect of the present invention relates to methods for treatmentof coronary heart disease comprising administering to a subject apeptide, compound, or mimetic of these or a pharmaceutical compositionof the present invention. In a preferred embodiment the subject is ahuman.

Yet another aspect of the present invention relates to methods fortreating or preventing inflammation in a subject comprisingadministering to the subject a peptide, compound, or mimetic of these ora pharmaceutical composition of the present invention. In a preferredembodiment the subject is a human.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a line graph of results from experiments examining the in vivoeffects of liposomes containing various SAA2.1 synthetic peptides onmacrophage cholesterol efflux. Results for liposomes containing thesynthetic peptide of amino acids 1 through 20 (SEQ ID NO:1) of mouseSAA2.1 are depicted as closed circles. Results for liposomes containingthe synthetic peptide of amino acids 21 through 50 (SEQ ID NO:2) ofmouse SAA2.1 are depicted as open circles. Results for liposomescontaining the synthetic peptide of amino acids 51 through 80 (SEQ IDNO:3) of mouse SAA2.1 are depicted as closed triangles. Results forliposomes containing the synthetic peptide of amino acids 74 through 103(SEQ ID NO:4) of mouse SAA2.1 are depicted as open triangles. Resultsfor liposomes containing the synthetic peptide of amino acids 1 through20 (SEQ ID NO:5) of mouse SAA1.1 are depicted as closed squares. Resultsfor liposomes containing the synthetic peptide of amino acids 1-23 (SEQID NO:6) of human SAA1.1 are depicted as open squares.

FIG. 2 is a line graph depicting cholesterol efflux in tissue culturemediated by L-form (SEQ ID NO:9) and D-form (SEQ ID NO:10) amino acidpeptides corresponding to residues 77-103 of murine SAA1.1. Cholesterolefflux following treatment with liposomes containing 0.5 :M cyanogenbromide-released peptides corresponding to amino acid residues 77-103 ofmurine SAA1.1 is depicted by open circles. Cholesterol efflux followingtreatment with liposomes containing synthetic D-form (SEQ ID NO:10)amino acid peptides of the corresponding sequence is depicted by closedtriangles. Cholesterol efflux following treatment with liposomescontaining the native L-form amino acid residues 74-103 of murine SAA2.1is depicted by inverted open triangles. Control, a chase efflux mediumconsisting of DMEM/BSA alone, is depicted as closed circles. The resultsrepresent cholesterol efflux to the acceptor, HDL, in the medium fromcells after pre-treatment with these liposomes. At various time points,the efflux media were collected and analyzed for [³H] cholesterol. Total[³H] cholesterol was (1.8-2.1)×10⁶ dpm/mg cell protein. Results are themean±SEM of four determinations.

FIGS. 3A and 3B are line graphs depicting dose-response data of an invitro cholesterol export study in macrophages administered liposomescontaining a peptide corresponding to amino acids 1-20 of murine SAA2.1(SEQ ID NO:1), liposomes containing a peptide corresponding to aminoacids 74-103 of murine SAA2.1 (SEQ ID NO:4), or a combination of thesepeptides (SEQ ID NO:1+SEQ ID NO:4) as liposomes in a 1:1 ratio.Concentrations of peptides examined include 0.05, 0.1, 0.5, 1.0, 2.5 and5.0 μM. In FIG. 3A, the X-axis, concentration, is depicted on a linearscale and in FIG. 3B the X-axis, concentration, is depicted on alogarithmic scale. As shown by these graphs, each of the peptides aloneincreased cholesterol efflux in response to increased dose. Further, acombination of the two peptides produced a greater than additive effect.

FIG. 4 is a line graph showing a time course of cholesterol efflux fromcholesterol-laden human THP-1 cells exposed to liposomal formulationscomprising various peptides of the present invention. Cholesterol effluxof these human cells following exposure to liposomes alone is depictedby filled circles. Cholesterol efflux of these human cells followingexposure to HDL is depicted by open circles. Cholesterol efflux of thesehuman cells following exposure to liposomes containing a peptidecorresponding to amino acids 1-20 of murine SAA2.1 (SEQ ID NO:1) isdepicted by filled triangles. Cholesterol efflux of these human cellsfollowing exposure to liposomes containing a peptide corresponding toamino acids 74-103 of murine SAA2.1 (SEQ ID NO:4) is depicted by opentriangles. Cholesterol efflux of these human cells following exposure toliposomes containing a peptide corresponding to amino acids 1-20 ofmurine SAA2.1 plus an arginine at the N-terminus (SEQ ID NO:7) isdepicted by filled squares. Cholesterol efflux of these human cellsfollowing exposure to liposomes containing a peptide corresponding toamino acids 1-20 of murine SAA2.1 (SEQ ID NO:1) and a peptidecorresponding to amino acids 74-103 (SEQ ID NO:4) of murine SAA2.1 isdepicted by open squares.

FIGS. 5A and 5B are line graphs depicting dose-response data of an invitro cholesterol export study in macrophages administered liposomescontaining a peptide corresponding to amino acids 78-96 of human SAA1.1(SEQ ID NO:12) and liposomes containing a peptide corresponding to aminoacids 74-103 of murine SAA2.1 (SEQ ID NO:4). Concentrations of peptidesexamined include 0.05, 0.1, 0.5, 1.0, 2.5 and 5.0 μM. In FIG. 5A, theX-axis, concentration, is depicted on a linear scale and in FIG. 5B, theX-axis, concentration, is depicted on a logarithmic scale. As shown bythese graphs, the human SAA1.1 peptide was at least as effective if notmore effective at increasing cholesterol efflux in response to increaseddose than the mouse peptide.

FIG. 6 is a line graph showing a time course of cholesterol efflux fromcholesterol-laden human THP-1 cells exposed to liposomal formulationscomprising various concentrations of the peptide of residues 78-96 ofhuman SAA1.1. Cholesterol efflux of these human cells following exposureto liposomes containing 0.05 μM hSAA1.1₇₈₋₉₆ is depicted as filledcircles. Cholesterol efflux of these human cells following exposure toliposomes containing 0.05 μM hSAA1.1₇₈₋₉₆ is depicted by filled circles.Cholesterol efflux of these human cells following exposure to liposomescontaining 0.1 μM hSAA1.1₇₈₋₉₆ is depicted by open circles. Cholesterolefflux of these human cells following exposure to liposomes containing0.5 μM hSAA1.1₇₈₋₉₆ is depicted by filled triangles. Cholesterol effluxof these human cells following exposure to liposomes containing 1.0 μMhSAA1.1₇₈₋₉₆ is depicted by open triangles. Cholesterol efflux of thesehuman cells following exposure to liposomes containing 2.5 μlhSAA1.1₇₈₋₉₆ is depicted by filled squares. Cholesterol efflux of thesehuman cells following exposure to liposomes containing 5.0 μlhSAA1.1₇₈₋₉₆ is depicted by open squares.

FIGS. 7A and 7B are bar graphs depicting the ability of liposomalformulations containing peptides of the present invention to reduce orcause regression of aortic lesions in ApoE knockout mice. FIG. 7B isinclusive of data presented in FIG. 7A as well as additional data from asubsequent experiment performed under the same conditions. In theseexperiments, animals were placed on an atherogenic diet (Paigen'sAtherogenic Rodent Diet: Purina 5015 with cocoa butter, cholesterol andcholic acid (CI3002, Research Diets, Inc.)) for four weeks, followingwhich they were divided into two groups. One group continued on the dietfor an additional two weeks. The other group continued on the diet forthe same period but also received once every four days liposomescontaining a peptide corresponding to amino acids 1-20 of murine SAA2.1(SEQ ID NO:1; Group B of FIG. 7A; hf+p1 of FIG. 7B) or liposomescontaining a peptide corresponding to amino acids 74-103 of murineSAA2.1 (SEQ ID NO:4; Group D of FIG. 7A; hf+p4 of FIG. 7B). The controlgroup received high fat diet alone with no liposomes (Group A of FIG.7A; high fat of FIG. 7B). An additional group was placed on a normalmouse chow diet (Group C of FIG. 7A; low fat of FIG. 7B). An additionalGroup receiving liposomes containing a peptide corresponding to aminoacids 1-20 of murine SAA2.1 (SEQ ID NO:1) and liposomes containing apeptide corresponding to amino acids 74-103 of murine SAA2.1 (SEQ IDNO:4) is depicted in FIG. 7B and is referred to as hf+p1+p4. After thetwo weeks, the mice were killed and their aortas were dissected andstained with Oil Red O. Data of FIG. 7A depict the area stained with OilRed O indicative of the actual lipid positive area or areas as apercentage of the total aortic area viewed. Data of FIG. 7B depict thearea stained with Oil Red O as a percentage relative to the high fatdiet group (100%). Five animals were used in each group in FIG. 7A. Thenumber of animals in each Group depicted in FIG. 7B is set forth as n.

FIGS. 8A and 8B are bar graphs the ability of liposomal formulationscontaining peptides of the present invention to prevent aortic lesionsin ApoE knockout mice. FIG. 8B is inclusive of data presented in FIG. 8Aas well as additional data from a subsequent experiment performed underthe same conditions. In the prevention experiments depicted in FIG. 8A,ApoE knockout mice were placed on a high fat diet and also received onceevery four days liposomes containing a peptide corresponding to aminoacids 1-20 of murine SAA2.1 (SEQ ID NO:1; Group 2), liposomes containinga peptide corresponding to amino acids 74-103 of murine SAA2.1 (SEQ IDNO:4; Group 4) or liposomes containing a peptide corresponding to aminoacids 1-20 of murine SAA2.1 and a peptide corresponding to amino acids74-103 of murine SAA2.1 (SEQ ID NO:1+SEQ ID NO:4; Group 5). The controlgroup received high fat diet alone with no liposomes (Group 1). Anadditional group was placed on a normal mouse chow diet (group 3). After20 days, the mice were killed and their aortas were dissected andstained with Oil Red O. Data of FIG. 8A depict the area stained with OilRed O indicative of actual lipid positive area or areas as a percentageof the total aortic area viewed. In FIG. 8A, five animals were used inGroups 1-3 and 5. Four animals were used in Group 4 as one animal diedduring the experiment. In FIG. 8B an additional experimental groupreferred to as “empty lipos” was included which are animals that weretreated with empty liposomes identical to those used in the peptidecontaining formulations but which are protein-peptide free. This groupis different from the high fat and low fat (diet) groups that were nottreated with liposomes. Data in FIG. 8B is expressed as the area stainedwith Oil Red O as a percentage relative to the high fat diet group(100%). In FIG. 8B the number of animals is set forth as n. Groupreferred to as “high fat”, “low fat”, “hf+p1”, “hf+p4” and hf+p(1+4)correspond to Groups 1, 3, 2, 4 and 5, respectively of FIG. 8A.

FIG. 9 is a line graph showing a time course study of in vitrocholesterol efflux in mouse J774 cells in the presence of liposomalformulations of various cholesterol ester hydrolase-enhancing peptidesof the present invention. Cholesterol efflux of cells in the presence ofa liposomal formulation containing human SAA1.1₇₈₋₉₆ (SEQ ID NO:12) isdepicted as filled circles. Cholesterol efflux of cells in the presenceof a liposomal formulation containing human SAA2.1₇₈₋₉₆ (SEQ ID NO:11)is depicted as open circles. Cholesterol efflux of cells in the presenceof a liposomal formulation containing human SAA2.1₇₉₋₉₆ (SEQ ID NO:26)is depicted as filled triangles. Cholesterol efflux of cells in thepresence of a liposomal formulation containing human SAA2.1₈₀₋₉₆ (SEQ IDNO:27) is depicted as open triangles. Cholesterol efflux of cells in thepresence of a liposomal formulation containing human SAA2.1₈₁₋₉₆ (SEQ IDNO:28) is depicted as filled squares.

FIG. 10 is a bar graph comparing the effects of equimolar concentrationsof liposomes containing full length murine SAA2.1 versus liposomescontaining a peptide corresponding to amino acids 1-20 of murine SAA2.1(SEQ ID NO:1 and amino acids 74-103 of murine SAA2.1 (SEQ ID NO:4) oncholesterol efflux in cholesterol-laden J774 cells.

DETAILED DESCRIPTION OF THE INVENTION

Approximately 13 million North Americans are taking cholesterol-loweringdrugs, and the majority of these individuals are now treated with thecategory of drugs known as statins. Cholesterol synthesis inhibitors(statins) are for the most part considered safe and highly effective.However, there have been some recent setbacks for this drug class. Forexample, the 2001 voluntary recall of Bayer's statin Baycol™, thedelayed North American introduction of AstraZeneca's statin Crestor™,and the recent concerns about the health risks associated with long-termstatin use (Clearfield, M. B., (2002) Expert Opin. Pharmacother.3:469-477) are indicative of the need for new drugs.

Thus, pharmaceutical companies are currently developing drugs that workvia different mechanisms from that of the current marketed drugs.Treatment with two or more drugs that act through different mechanismscan, in fact, be additive or synergistic in their combined ability toreduce cholesterol levels (Brown, W. V. (2001) Am. J. Cardiol. 87(5A):23B-27B; Buckert, E. (2002) Cardiology 97: 59-66). Ezetimibe (Zetia™,Merck), which was recently approved by the FDA, can significantly reducecholesterol levels by itself. Furthermore, since Ezetimibe works bydecreasing cholesterol absorption (i.e. blocks cholesterol transport),it can also be given with cholesterol synthesis inhibitors (statins) todecrease plasma cholesterol levels to a greater extent than when eitherdrug is given alone (Davis et al. (2001) Arterioscler Thromb Vasc Biol.21: 2031-2038; Rader, D. J. (2002) Am. J. Managed Care 8 (2 Supply:S40-44).

Other drugs such as Avasimibe (Pfizer), Eflucimibe (Eli Lilly) andCS-505 (Sankyo), which are in clinical trials, are aimed at inhibitingacyl CoA:cholesterol acyl transferase (ACAT) activity.

Companies such as Esperion Therapeutics, Tularik Inc. and the Canadiancompany, Xenon Genetics are developing ways to increase the levels ofHDL, the so-called “good cholesterol”, which plays a key role in thereverse cholesterol transport pathway, known to be important for theexcretion of cholesterol out of the body.

However, while there has been considerable effort by pharmaceuticalcompanies to produce new compounds for treating atherosclerosis, thereare currently no drugs on the market that have the ability to promotethe mobilization and efflux of stored cholesterol from macrophageslocated in atherosclerotic plaques by enhancing cholesterol esterhydrolase activity.

The accumulation of cholesterol in vascular cells such as macrophages isa defining pathological feature of atherosclerosis. Macrophages are keycells in the storage and removal of lipids. Their conversion to foamcells (cholesterol-laden macrophages) is an early and importantpathological process in the formation of an atherosclerotic plaque.

Two enzymes are critical for maintaining cellular cholesterol balance.

Cholesterol ester hydrolase, also referred to as cholesterol esteraseand cholesteryl ester hydrolase, promotes the removal or efflux ofcholesterol from macrophages. This enzyme exists in an acidic as well asa neutral form and all aspects of the present invention are applicableto both forms, with modulation of the neutral form being preferred.

Acyl CoA:cholesterol acyl transferase promotes the storage of macrophagecholesterol. During an acute phase inflammatory response, serum amyloidA (SAA) isoforms 1.1 and 2.1 become major constituents of high densitylipoprotein and this complex is internalized by macrophages. As shownherein, murine SAA2.1, but not murine SAA1.1, inhibits acylCoA:cholesterol acyl transferase activity and enhances cholesterol esterhydrolase activity, shifting the balance to the transportable form ofcholesterol. Serum amyloid A (SAA) has been demonstrated to have aspecific binding affinity for macrophages, separate from apoA-1 bindingaffinity for macrophages. Murine isoform 2.1 is the first protein shownto both enhance cholesterol ester hydrolase activity and inhibit acylCoA:cholesterol acyl transferase activity. However, as evidenced herein,human SAA1.1 and human SAA2.1 comprise peptide domains that enhancecholesterol ester hydrolase activity and inhibit acyl CoA:cholesterolacyl transferase activity.

The in vitro effects of acute phase-HDL (AP-HDL;HDL-SAA) on acylCoA:cholesterol acyl transferase and cholesterol ester hydrolaseactivities and on cellular cholesterol export were studied by theinventors using purified enzymes, cell homogenates, and whole cells.Results from in vitro studies using macrophage post-nuclear homogenatesas a source of acyl CoA:cholesterol acyl transferase showed murineSAA2.1 to inhibit acyl CoA:cholesterol acyl transferase activity in adose-dependent manner. In contrast, murine SAA1.1 and apoA-1 had noeffect. AP-HDL, as well as liposomes containing murine SAA2.1, were alsoshown by the inventors to cause a marked reduction of acylCoA:cholesterol acyl transferase activity and enhancement of cholesterolester hydrolase activity in intact cholesterol-laden macrophages intissue culture. In contrast, HDL alone, SAA2.1-free liposomes, andliposomes containing murine SAA1.1 or apoA-1 had no effect on enzymeactivities. Using macrophages preloaded with radio-labeled cholesteroland injected intravenously into either inflamed or un-inflamed mice, itwas shown that the inflamed mice, which have high SAA2.1 levels,effluxed cholesterol more rapidly and to a greater extent (6-foldgreater) than their un-inflamed counterparts. Further, usingcholesterol-loaded macrophages pretreated with liposomes containingeither murine SAA1.1, murine SAA2.1, or apoA-1 and then injectedintravenously into un-inflamed mice, the inventors have now shown thatonly liposomes containing murine isoform 2.1 recapitulated the majorcholesterol releasing effect seen in inflamed mice.

Using both in vitro and in vivo assays, these unique properties ofmurine SAA2.1 have been demonstrated to reside in two peptide domains.The acyl CoA:cholesterol acyl transferase inhibitory domain of murineSAA2.1 resides in residues 1-16 of the N-terminus of SAA2.1. ThisN-terminal 16-residue domain released by cyanogen bromide cleavage ofmurine SAA2.1, produced no effect, however, on cholesterol esterhydrolase activity. Instead, the C-terminal 30-residue domain of murineSAA2.1 correlating to amino acids 74-103 of murine SAA2.1 has now beenidentified as the region of murine SAA2.1 that enhances cholesterolester hydrolase activity. In particular, the cholesterol ester hydrolaseactivity-enhancing domain has been identified as correlating to aminoacids 77-95 of murine SAA2.1.

As shown herein, isolated peptides with amino acid sequences comprisingthese domains within murine SAA2.1 and human SAA1.1 and human SAA2.1have a potent enhancing effect on macrophage cholesterol ester hydrolaseactivity and an inhibiting effect on acyl CoA:cholesterol acyltransferase activity both in vitro and in vivo. Peptides synthesized tocontain the amino acid sequences of these domains or portions thereofhave the ability to shift macrophage cholesterol into a transportableform that is then rapidly exported from the cell in the presence of afunctional cholesterol transporter and cholesterol acceptor high densitylipoprotein. Further, these isolated peptides are extremely active, as asingle intravenous injection mobilizes macrophage cholesterol in vivofor over 4 days.

Peptides corresponding to amino acid residues 1-20(GFFSFVHEAFQGAGDMWRAY; SEQ ID NO:1), 21-50(TDMKEANWKNSDKYFHARGNYDAAQRGPGG; SEQ ID NO:2), 51-80(VWAAEKISDGREAFQEFFGRGHEDTIADQE; SEQ ID NO:3) and 74-103(DTIADQEANRHGRSGKDPNYYRPPGLPDKY; SEQ ID NO:4) of murine SAA2.1 proteinsequence, respectively, were synthesized by solid-phase peptidesynthesis. A peptide corresponding to amino acid residues 1-23(RSFFSFLGEAFDGARDMWRAYSD; SEQ ID NO:6) of human SAA1.1 and/or humanSAA2.1 was also synthesized as well as peptides corresponding toresidues 78-96 of human SAA2.1 (ADQAANKWGRSGRDPNHFR; SEQ ID NO:11),residues 79-96 of human SAA2.1 (DQAANKWGRSGRDPNHFR; SEQ ID NO:26),residues 80-96 of human SAA2.1 (QAANKWGRSGRDPNHFR; SEQ ID NO:27) andresidues 81-96 of human SAA2.1 (AANKWGRSGRDPNHFR; SEQ ID NO:28). Inaddition, a peptide corresponding to amino acid residues 1-20(GFFSFIGEAFQGAGDMWRAY; SEQ ID NO:5) of murine SAA1.1 protein sequencewas synthesized, as well as a peptide corresponding to amino acidresidues 1-20 of murine SAA1.1 protein sequence plus an arginine at theN-terminus (RGFFSFIGEAFQGAGDMWRAY; SEQ ID NO:7). Synthetic peptides SEQID NO: 1 through 7 comprise L amino acids. These synthetic peptides ofthe present invention are nonglycosylated, as are the native forms ofSAA1.1 and SAA2.1.

Further, cyanogen bromide cleavage of murine SAA2.1 has been shown togenerate an insoluble 16-mer (SAA2.1₁₋₁₆) and two soluble fragments, a7-mer (SAA2.1₁₇₋₂₃) and an 80-mer (SAA2.1₂₄₋₁₀₃; depicted herein as SEQID NO:23) (Ancsin, J. et al. J. Biol. Chem. 274: 7172-7181, 1999). Formurine SAA1.1, a substitution of Ile with Met at residue 76 introducesan additional cleavage site, allowing the 80-mer to be cleaved into a53-mer (SAA1.1₂₄₋₇₆) and a 27-mer (SAA1.1₇₇₋₁₀₃). The last 27 residuesof murine SAA2.1 and SAA1.1 are as follows:

SAA2.1 ₇₇₋₁₀₃ ADQEANRHGRSGKDPNYYRPPGLP D KY (SEQ ID NO: 8) SAA1.1 ₇₇₋₁₀₃ADQEANRHGRSGKDPNYYRPPGLP A KY (SEQ ID NO: 9)The only difference in amino acid residues in these two sequencesresides at position 101 (bold and underlined).

These synthetic peptides were used to map a domain in SAA2.1 that isresponsible for modulating cholesterol ester hydrolase and to identifycompositions useful in modulating cholesterol ester hydrolase activityand/or acyl CoA:cholesterol acyl transferase activity.

As shown herein, pre-incubation of J774 macrophages with liposomescontaining 0.5 μM synthetic peptide corresponding to amino acid residues74-103 of murine SAA2.1 (SEQ ID NO:4) resulted in a significant increasein the rate of macrophage [³H] cholesterol efflux into medium containingHDL. Liposomes containing a shorter peptide generated from the CNBrcleavage of native murine SAA1.1 protein (SAA1.1₇₇₋₁₀₃; SEQ ID NO:9)have also now been found by the inventors to have a similar effect.Specifically, [³H] cholesterol efflux into the medium was demonstratedto be similar in both murine SAA1.1₇₇₋₁₀₃-treated and murineSAA2.1₇₄₋₁₀₃-treated macrophages.

These data indicate that residues 74-76 of murine SAA may not benecessary in promoting macrophage cholesterol efflux. Further, residue101 is believed to be unnecessary. In fact, an examination of thesequences of approximately 12 species indicates that the terminal 8residues of SAA1.1 and 2.1, which are rich in proline, are likelyunnecessary for CEH enhancing activity. Accordingly, it is believed thata peptide of 19 residues from amino acids 77 through 95 of murine SAA2.1possesses the CEH enhancing property. Further, a peptide comprising theconsensus sequence ADQAANEWGRSGKDPNHFR (SEQ ID NO:12) corresponding toresidues 78 through 96 of human SAA1.1 and peptides corresponding toresidues 78 through 96 of human SAA2.1 (ADQAANKWGRSGRDPNHFR; SEQ IDNO:11) and residues 79 through 96 of human SAA2.1 (DQAANKWGRSGRDPNHFR;SEQ ID NO:26) are shown herein to increase export of cholesterol. Thus,this peptide and peptides corresponding to residues through 96 of humanSAA2.1 (ADQAANKWGRSGRDPNHFR; SEQ ID NO:11) and residues 79 through 96 ofhuman SAA2.1 (DQAANKWGRSGRDPNHFR; SEQ ID NO:26) are believed to possessCEH enhancing activity as well.

Additionally, these data further elucidate the differences between thetertiary structures (i.e., 3-dimensional structures, protein folding) ofSAA1.1 and SAA2.1, since, unlike SAA2.1, the native SAA1.1 protein doesnot promote macrophage cholesterol efflux. This information andadditional modeling work is useful in the molecular modeling of the SAAprotein and peptides for the design of small molecule mimetics.

Modifications of such peptides to comprise one or more D amino acidswere also shown by the inventors to result in equally effective peptidesexpected to be more stable and less susceptible to degradation in vivo.See FIG. 2. A synthetic peptide corresponding to amino acid sequence77-103 of murine SAA1.1 which consists of all D-amino acids (D-formADQEANRHGRSGKDPNYYRPPGLPAKY, referred to herein as SEQ ID NO:10), had asimilar effect in enhancing macrophage cholesterol export into themedium when cells were treated in parallel with the corresponding nativeL-amino acid peptide of murine SAA1.1.

Identification of the domain of SAA2.1 that is responsible for enhancingcholesterol ester hydrolase activity was performed in J774 cellspreloaded with radio-labeled cholesteryl esters. These experiments wereperformed in the presence of Sandoz 58-035, an inhibitor of acylCoA:cholesterol acyl transferase activity, to prevent re-esterificationof liberated cholesterol and [¹⁴C]oleate. Incubations proceeded fortimes ranging from 0 to 24 hours, following which the remainingquantities of [¹⁴C]-labeled labeled cholesteryl oleate in cells weremeasured to determine the rate of hydrolysis of cholesteryl ester. Withre-esterification blocked, there were no significant differences in therate of hydrolysis of [¹⁴C]-labeled cholesteryl oleate in cells culturedin the presence of protein-free liposomes or liposomes containing 0.5 μMsynthetic peptides corresponding to amino acid residues 1-20, 21-50 and51-80 of murine SAA2.1, respectively. However, an equivalent amount ofliposomes containing the synthetic peptide corresponding to amino acidresidues 74-103 of murine SAA2.1 caused a 3-fold increase in cholesterolester hydrolase activity in these cholesterol-laden murine cells.

The incorporation of [¹⁴C]oleate into cholesteryl ester was used as ameasure of acyl CoA:cholesterol acyl transferase activity to identifycompositions inhibiting the enzyme activity. The relative acylCoA:cholesterol acyl transferase activity was determined incholesterol-laden murine cells that had been cultured in medium in theabsence of liposomes or in the presence of protein-free liposomes orliposomes containing 0.5 μM synthetic peptides corresponding to aminoacids 1-20, 21-50, 51-80 and 74-103 of murine SAA2.1. Following a 6 hourincubation, only the cells that had been exposed to liposomes containingsynthetic peptides corresponding to amino acid residues 1-20 of murineSAA2.1 showed a two-fold decrease in acyl CoA:cholesterol acyltransferase activity, while other liposome treatments had no significanteffect on the activity of this enzyme.

Cholesterol efflux from cholesterol-loaded J774 cells pre-incubated withliposomes containing one of the above synthetic peptides of murineSAA2.1 was also examined. In these experiments, cholesterol-loadedmurine macrophages labeled with [³H]cholesterol were pre-incubated for 4hours with liposomes containing 0.5 μM synthetic peptides correspondingto amino acids 1-20, 21-50, 51-80 or 74-103 of murine SAA2.1. In someexperiments, an equimolar combination of two synthetic peptides (0.5 μMeach) corresponding to amino acid residues 1-20 and 74-103 of murineSAA2.1 was also assayed. Following incubation, the cells were washedextensively with DMEM/BSA to remove all radioactivity and liposomes inthe pre-incubation medium. The chase efflux consisted of DMEM/BSA aloneor medium containing HDL (50 μg/mL). At various time points, the effluxmedia were collected and analyzed for [³H]cholesterol and freecholesterol mass. Results indicated that [³H]cholesterol efflux tomedium containing 0.2% BSA was 6.1±1.1% of total counts. Cells culturedin the presence of HDL (50 μg/mL), exported 31.2±2.2% of total cellular[³H] sterol to the medium. Pre-incubation of cells with liposomescontaining 0.5 μM synthetic peptides corresponding to amino acidresidues 21-50 or 51-80 of murine SAA2.1 did not cause any significantchanges in the rate of [³H]cholesterol efflux into the medium containingthe HDL. However, when cholesterol-laden J774 cells labeled with[³H]cholesterol were pre-incubated with liposomes containing 0.5 μMsynthetic peptides corresponding to amino acid residues 1-20 or 74-103of murine SAA2.1, it was observed that 60.6±3.6% and 46.7±3.1% of totalcellular [³H]cholesterol were released into the medium when cells weresubsequently cultured in the presence of HDL. Under similar culturingconditions, pre-incubation with the combination of these two syntheticpeptides of SAA2.1 resulted in the export of 88.5±3.5% of total cellular[³H]cholesterol to HDL. In addition, the initial rate of cholesterolefflux to HDL during the first 2 hours was twice as fast when comparedto the results with liposomes containing either synthetic peptide alone.

Further, as shown in FIG. 10, comparison of cholesterol efflux byequimolar concentrations of liposomes containing the full length murineSAA2.1 protein and liposomes containing synthetic peptides correspondingto amino acid residues 1-20 or 74-103 of murine SAA2.1 showed astatistically significant greater cholesterol efflux fromcholesterol-laden J744 cells receiving liposomes containing syntheticpeptides corresponding to amino acid residues 1-20 or 74-103 of murineSAA2.1.

Dose-response curves of cholesterol export in cholesterol-laden murinemacrophages were also generated for liposomes containing a peptidecorresponding to amino acids 1-20 of murine SAA2.1 (SEQ ID NO:1),liposomes containing a peptide corresponding to amino acids 74-103 ofmurine SAA2.1 (SEQ ID NO:4) and liposomes containing a combination ofthese peptides (SEQ ID NO:1+SEQ ID NO:4) in a 1:1 molar ratio. Resultsare depicted in FIGS. 3A and 3B. Concentrations of peptides examinedincluded 0.05, 0.1, 0.5, 1.0, 2.5 and 5.0 μM. Each of the peptides aloneincreased cholesterol efflux and the percent of cholesterol efflux ascompared to controls increased with increasing amounts of each peptidealone. Further, as shown in FIGS. 3A and 3B, the combination of the twopeptides produced a greater than additive effect. For example, as shownin FIGS. 3A and 3B, cholesterol efflux with 1 μM of the peptides alonewas approximately 200% while cholesterol efflux with the combination ofpeptides at 1 μM was 500%.

These peptides have also now been demonstrated to increase cholesterolefflux in human derived monocytic cells. These monocytes weredifferentiated into macrophages with phorbol myristate acetate (100 nM).In these experiments, cholesterol-laden human THP-1 cells were exposedto liposomal formulations comprising a peptide corresponding to aminoacids 1-20 of murine SAA2.1 (SEQ ID NO:1), a peptide corresponding toamino acids 74-103 of murine SAA2.1 (SEQ ID NO:4), a peptidecorresponding to amino acids 1-20 of murine SAA1.1 plus an arginine atthe N-terminus (SEQ ID NO:7), or a peptide corresponding to amino acids1-20 of murine SAA2.1 (SEQ ID NO:1) and a peptide corresponding to aminoacids 74-103 (SEQ ID NO:4) of murine SAA2.1. Results from theseexperiments are depicted in FIG. 4. Unlike the peptide corresponding toamino acids 1-20 of murine 1.1 (SEQ ID NO:5), which is inactive, theliposomal formulation containing peptide corresponding to amino acids1-20 of murine SAA2.1 plus an arginine at the N-terminus (SEQ ID NO:7)effectively increased cholesterol efflux in these human cells equal to,if not better than, peptides corresponding to amino acids 1-20 of murineSAA2.1 (SEQ ID NO:1) and amino acids 74-103 of murine SAA2.1 (SEQ IDNO:4).

Thus, the rate of cholesterol export may increase with differentpeptides and/or with increasing concentrations of a peptide.

Similar studies were performed with the peptide corresponding toresidues 78-96 of human SAA1.1 (ADQAANEWGRSGKDPNHFR; SEQ ID NO:12).FIGS. 5A and 5B show dose response curves from an in vitro cholesterolexport study in macrophages administered liposomes containing a peptidecorresponding to amino acids 78-96 of human SAA1.1 (SEQ ID NO:12) orliposomes containing a peptide corresponding to amino acids 74-103 ofmurine SAA2.1 (SEQ ID NO:4). Concentrations of peptides examined were0.05, 0.1, 0.5, 1.0, 2.5 and 5.0 μM. As shown by FIGS. 5A and 5B, thehuman SAA1.1 peptide (SEQ ID NO:12) was at least as effective if notmore effective at increasing cholesterol efflux in response to increaseddose than the mouse peptide. Accordingly, these results are indicativeof human SAA1.1 peptides comprising SEQ ID NO:12 being enhancers ofcholesterol ester hydrolase activity.

FIG. 9 shows a time course study of cholesterol efflux in mouse 774cells with liposomal formulations containing human SAA1.1₇₈₋₉₆ (SEQ IDNO:12), human SAA2.1₇₈₋₉₆ (SEQ ID NO:11), human SAA2.1₇₉₋₉₆ (SEQ IDNO:26), human SAA2.1₈₀₋₉₆ (SEQ ID NO:27) or human SAA2.1₈₁₋₉₆ (SEQ IDNO:28). As shown therein, the liposomal formulation containing humanSAA1.1₇₈₋₉₆ (SEQ ID NO:12) exhibited the greatest cholesterol exportenhancing activity of the formulations examined. Liposomal formulationscontaining human SAA2.1₇₈₋₉₆ (SEQ ID NO:11) or human SAA2.1₇₉₋₉₆ (SEQ IDNO:26) also exhibited cholesterol export enhancing activity with eachhaving an activity of about half of the human SAA1.1₇₈₋₉₆ containingliposomal formulation. Liposomal formulations containing humanSAA2.1₈₀₋₉₆ (SEQ ID NO:27) or human SAA2.1₈₁₋₉₆ (SEQ ID NO:28) exhibitedlittle to no cholesterol export enhancing activity thus indicating thepresence of at least residue 79 to be important to the activity of thesepeptides.

A time course of cholesterol efflux was also performed incholesterol-laden human THP-1 cells exposed to liposomal formulationscomprising various concentrations of a peptide corresponding to residues78-96 of human SAA1.1. Results from this experiment are depicted in FIG.6. As shown therein, cholesterol efflux from macrophages continued toincrease over a time period from 0 to 24 hours for all concentrations ofpeptide examined.

Additional in vivo studies have also been conducted wherein mice werefirst injected intravenously with [³H]cholesterol-laden macrophages andthen injected 24 hours later with liposomes containing 0.5 μM ofsynthetic peptides corresponding to amino acid residues 1-20, 21-50,51-80 or 74-103 of murine SAA2.1. Results from this study are depictedin FIG. 1. At time points indicated in the graph of FIG. 1,approximately 25 μl of blood were collected from the tail vein of eachanimal. The blood samples were centrifuged to separate the red bloodcells from the plasma and the [³H]-cholesterol in plasma was determinedby scintillation counting. Results are mean±SEM of four determinations.As shown in this Figure, intravenous injection of liposomes containingeither SAA2.1 peptide residues 1-20 (acyl CoA:cholesterol acyltransferase-inhibiting domain), or residues 74-103 (cholesterolhydrolase ester-enhancing domain) dramatically increased[³H]-cholesterol efflux as measured by an increase in plasmaradioactivity (dpm). In the same study, the human acyl CoA:cholesterolacyl transferase-inhibiting SAA1.1 peptide domain, which is equivalentto the human and mouse SAA2.1 domain, also promoted in vivo cholesterolexport.

These experiments demonstrate that liposomes containing native SAA2.1protein, or synthetic peptides comprising the murine acylCoA:cholesterol acyl transferase-inhibiting domain or the murine orhuman cholesterol ester hydrolase enhancing domain, markedly increasedin vivo cholesterol efflux. Further, this increase lasted for over 4days. Additionally, the human acyl CoA:cholesterol acyltransferase-inhibiting SAA peptide domain also promoted in vivocholesterol efflux. Thus, these data are demonstrative of the key roleSAA, and in particular the acyl CoA:cholesterol acyltransferase-inhibiting domain and the cholesterol hydrolaseester-enhancing domain of this protein, play in facilitating cholesterolremoval from cholesterol-laden macrophages located in atheroscleroticplaques. The data substantiate the utility of designing and usingpeptides or mimetics of these domains to reduce or prevent atherogenesisand/or cause regression of an atherosclerotic plaque by increasing theefflux of cholesterol from macrophages located in an atheroscleroticlesion. Such peptides or mimetics thereof will be useful in thetreatment or prevention of atherosclerosis and in the treatment ofcoronary heart disease and cardiovascular disease associated withatherosclerosis.

The export process of cholesterol is coupled to the ATP binding cassettetransporter (ABCA1) pathway. Lipid efflux to apolipoproteins has beenshown previously to be stimulated by treatment of murine macrophageswith cAMP analogues (Lin et al. 2002 Biochem Biophys Res. Commun.290:663-669; Oram et al. 2000 J. Biol. Chem. 275:34508-34511). Also, theexpression of ABCA1 is induced by cAMP treatment (Lin et al. 2002Biochem Biophys Res. Commun. 290:663-669; Oram et al. 2000 J. Biol.Chem. 275:34508-34511). The inventors herein examined the effect of8-bromo-cAMP (0.3 mM) on cholesterol efflux by liposomes containingvarious apolipoproteins when incubated with cholesterol-laden J774macrophages. Such cells were pre-labeled with [³H]-cholesterol in thepresence of Sandoz 58-035, an ACAT inhibitor, to ensure that all of theradiolabeled cholesterol released from the cells was derived from theun-esterified cholesterol pool, and the cells were treated for 12 hourswith 8-bromo-cAMP. This was followed by incubation with variousacceptors. The fractional release of cellular labeled cholesterol wasdetermined as a function of time. When compared to untreated cells, cAMPpre-treatment resulted in a 62.1% and 32.7% increase in the initial rateof cholesterol efflux to liposomes containing SAA2.1 and apoA-1. Nostimulation of efflux was observed when cells were exposed to SAA1.1liposomes with or without cAMP pre-treatment. Furthermore, it has beendemonstrated previously that SAA1.1 liposomes are not any more effectivethan protein-free liposomes in promoting cholesterol efflux fromcholesterol-laden macrophages (Tam et al. 2002 J. Lipid Res.43:1410-1420). Moreover, cAMP treatment did not stimulate cholesterolexport to culture medium containing no liposomes.

To investigate whether an apolipoprotein-free acceptor such ascyclodextrin has the ability to catalyze the removal of cholesterol frommacrophages, cholesterol-loaded and labeled J774 cells were incubatedwith liposomes and methyl-β-cyclodextrin (0.1 mM) (CD). No stimulationof cholesterol efflux to medium containing no liposomes was observed atthis concentration of CD. In contrast, CD treatment resulted in a 4-foldincrease in the initial rate of cholesterol efflux in cells treated withliposomes containing SAA2.1, but not liposomes containing SAA1.1, norprotein-free liposomes. Furthermore, cAMP pre-treatment caused a furtherincrease (45.5%) in cholesterol efflux in cells exposed to liposomescontaining SAA2.1 and CD.

Thus, the present invention provides isolated peptides, Y—Z and Q-Y—Zcompounds and mimetics of these, and pharmaceutical compositionscomprising an isolated peptide or portion thereof, a Y—Z or Q-Y—Zcompound or a mimetic of these, for use in the prevention and/ortreatment of atherosclerosis as well as coronary heart disease andcardiovascular disease associated with atherosclerosis. Pharmaceuticalcompositions of the present invention comprise a peptide or portionthereof, a Y—Z or Q-Y—Z compound, or a mimetic of these, of thecholesterol ester hydrolase enhancing domain of SAA2.1; and/or a peptideor portion thereof, a Y—Z or Q-Y—Z compound, or a mimetic of these, ofthe acyl CoA:cholesterol acyl transferase inhibitory domain of SAA2.1.Thus, preferred compositions of the present invention comprise a peptidecontaining amino acids 77-95 of mouse SAA2.1 or 78-96 of human SAA1.1 ora portion thereof, and/or a peptide containing residues 1-16 of SAA2.1or a portion thereof, or a mimetic of either or both of these peptidesor portions thereof.

By “portion thereof” it is meant to be inclusive of peptides exhibitingsimilar biological activities to the isolated peptides described hereinbut which, (1) comprise shorter fragments of the 19 residue cholesterolester hydrolase enhancing domain or the 16 residue acyl CoA:cholesterolacyl transferase inhibitory domain of murine SAA2.1 or human SAA1.1 orSAA2.1, or (2) overlap with only part of the 19 residue cholesterolenhancing domain or the 16 residue acyl CoA:cholesterol acyl transferaseinhibitory domain of murine SAA2.1 or human SAA1.1 or SAA2.1. Forexample, it is believed that peptides comprising the portion of the acylCoA:cholesterol acyl transferase inhibitory domain of murine SAA2.1 orhuman SAA1.1 or SAA2.1 extending from residues about 1 to 12, 1 to 13,or 1 to 14 will also inhibit acyl CoA:cholesterol acyl transferasesimilarly to the synthetic peptides of residues 1-20 of murine SAA2.1and residues 1-23 of human SAA1.1 and SAA2.1. Similarly, a preferredportion of the 30 amino acid sequence of residues 74-103 of murineSAA2.1 with cholesterol ester hydrolase enhancing activity has beenidentified and comprises a 19 amino acid region corresponding toresidues 77 through 95 of this domain. Similarly, 18 to 19 amino acidregions corresponding to residues 79 through 96 or 78 through 96,respectively, of human SAA1.1 have been identified and demonstrated tohave cholesterol ester enhancing properties. Shorter portions of these77-95, 78-96 or 79-96 residue peptides with similar biologicalactivities can be identified in the same manner as these 77-95, 78-96 or79-96 residue peptides. Accordingly, the present invention relates toportions of the peptides taught herein as well.

A preferred peptide of the present invention is the synthetic peptidecorresponding to amino acid residues 1-20 of murine SAA1.1 proteinsequence plus an arginine at the N-terminus (RGFFSFIGEAFQGAGDMWRAY; SEQID NO:7).

By synthetic, as used herein it is meant that the peptide is preparedsynthetically either by chemical means or recombinantly.

Further, it will of course be understood, without the intention of beinglimited thereby, that a variety of substitutions of amino acids in thedisclosed peptides is possible while preserving the structureresponsible for the cholesterol ester hydrolase enhancing activity orthe acyl CoA:cholesterol acyl transferase inhibitory activity of thepeptides disclosed herein. Conservative substitutions are described inthe patent literature, as for example, in U.S. Pat. No. 5,264,558. It isthus expected, for example, that interchange among non-polar aliphaticneutral amino acids, glycine, alanine, proline, valine and isoleucine,would be possible. Likewise, substitutions among the polar aliphaticneutral amino acids, serine, threonine, methionine, asparagine andglutamine could possibly be made. Substitutions among the charged acidicamino acids, aspartic acid and glutamic acid, could possibly be made, ascould substitutions among the charged basic amino acids, lysine andarginine. Substitutions among the aromatic amino acids, includingphenylalanine, histidine, tryptophan and tyrosine would also likely bepossible. In some situations, histidine and basic amino acids lysine andarginine may be substituted for each other. These sorts of substitutionsand interchanges are well known to those skilled in the art. Othersubstitutions might well be possible. It is expected that the greaterthe percentage of sequence identity of a variant peptide with a peptidedescribed herein, the greater the retention of biological activity.Accordingly, peptide variants having the activity of enhancingcholesterol ester hydrolase and/or inhibiting acyl CoA:cholesterol acyltransferase as described herein are encompassed within the scope of thisinvention.

Preferred for use in the present invention is an isolated peptide(X)_(n)FFX₁FX₂X₃X₄X₅FX₆ or a portion thereof wherein F is phenylalanineor an amino acid which is a conservative substitution thereof and n is 1or 2. Thus when n is 1, the isolated peptide comprises XFFX₁FX₂X₃X₄X₅FX₆(SEQ ID NO:13) wherein F is phenylalanine or an amino acid which is aconservative substitution thereof, X, X₁, X₄, X₅ and X₆ areindependently any amino acid, X₂ is a hydrophobic or nonpolar aminoacid; and X₃ is histidine or an amino acid which is a conservativesubstitution thereof. When n is 2, the isolated peptide comprisesX_(a)X_(b)FFX₁FX₂X₃X₄X₅FX₆ (SEQ ID NO:14), wherein F is phenylalanine oran amino acid which is a conservative substitution thereof, X_(a) and X₆are amino acids capable of forming a salt bridge, and X_(b), X, X₁, X₂,X₃, X₄ and X₅ are independently any amino acid, or a mimetic thereof.Examples of amino acid combinations of X_(a) and X₆ forming salt bridgesinclude, but are not limited to, X_(a) being arginine and X₆ beingaspartic acid or glycine. More preferred are isolated peptidesconsisting of amino acid residues 1-20 (GFFSFVHEAFQGAGDMWRAY SEQ IDNO:1) of murine SAA2.1, amino acid residues 1-23(RSFFSFLGEAFDGARDMWRAYSD; SEQ ID NO:6) of human SAA1.1 or SAA2.1, andRGFFSFIGEAFQGAGDMWRAY (SEQ ID NO:7). These isolated peptides of thepresent invention are capable of inhibiting acyl CoA:cholesterol acyltransferase. Excluded from the scope of the peptides of the presentinvention capable of inhibiting acyl CoA:cholesterol acyl transferaseactivity are those isolated peptides consisting of GFFSFVHEAFQGAGDM (SEQID NO:15), GFFSFIGEAFQGAGDM (SEQ ID NO:16), RSFFSFLGEAFDGARDMW (SEQ IDNO:17), GFFSFIGEAFQGAGDMWRAYTDMKEAGWKDGDKYFHARGNYDAAQRGPGGVWAAEKISDARESFQEFFGRGHEDTMADQEANRHGRSGKDPNYYRPPGLPAKY (full length murine SAA1.1;SEQ ID NO:18);GFFSFVHEAFQGAGDMWRAYTDMKEANWKNSDKYFHARGNYDAAQRGPGGVWAAEKISDGREAFQEFFGRGHEDTIADQEANRHGRSGKDPNYYRPPGLPDKY (full length murine SAA2.1;SEQ ID NO:19);RSFFSFLGEAFDGARDMWRAYSDMREANYIGSDKYFHARGNYDAAKRGPGGVWAAEAISDARENIQRFFGHGAEDSLADQAANEWGRSGKDPNHFRPAGLPEKY (full length human SAA1.1;SEQ ID NO:20); orRSFFSFLGEAFDGARDMWRAYSDMREANYIGSDKYFHARGNYDAAKRGPGGAWAAEVISNARENIQRLTGHGAEDSLADQAANKWGRSGRDPNHFRPAGLPEKY (full length human SAA2.1;SEQ ID NO:21).

Preferred isolated peptides capable of enhancing cholesterol esterhydrolase activity for use in the present invention compriseX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁X₁₂X₁₃X₁₄X₁₅X₁₆X₁₇X₁₈ (SEQ ID NO:29) or aportion thereof wherein X₁ and X₉, X₁₂ or X₁₈ are amino acids capable offorming a salt bridge, X₆ is glutamic acid or lysine or an amino acidwhich is a conservative substitution thereof, and X₂, X₃, X₄, X₅, X₇,X₈, X₁₀, X₁₁, X₁₃, X₁₄, X₁₅, X₁₆, and X₁₇ are independently any aminoacid. Preferred is the peptide comprisingX₁X₂X₃X₄X₅X₈X₇X₈X₉X₁₀X₁₁X₁₂X₁₃X₁₄X₁₅X₁₆X₁₇X₁₈ (SEQ ID NO: 29) wherein X₁and X₉, X₁₂ or X₁₈ are amino acids capable of forming a salt bridge, X₂is glutamine or an amino acid which is a conservative substitutionthereof, X₃ and X₄ are independently alanine or an amino acid which is aconservative substitution thereof, X₅ and X₁₅ are independentlyasparagine or an amino acid which is a conservative substitutionthereof, X₇ is tryptophan or an amino acid which is a conservativesubstitution thereof, X₈ and X₁₁ are independently glycine or an aminoacid which is a conservative substitution thereof, X₁₀ is serine or anamino acid which is a conservative substitution thereof, X₁₃ is asparticacid or an amino acid which is a conservative substitution thereof, X₁₄is proline or an amino acid which is a conservative substitutionthereof, X₁₆ is histidine or an amino acid which is a conservativesubstitution thereof, and/or X₁₇ is phenylalanine or an amino acid whichis a conservative substitution thereof. Examples of amino acidcombinations capable of forming a salt bridge include X₁ being anaspartic acid and X₉, X₁₂ or X₁₈ being an arginine. It is preferred thatthe isolated peptide or mimetic has less than 80 amino acid residues,more preferably 18 to 79 amino acids, more preferably 18 to 50 aminoacids, more preferably 18 to 35, 18 to 30, or 18 to 25 amino acids. Alsopreferred are isolated peptides DTIADQEANRHGRSGKDPNYYRPPGLPDKY (SEQ IDNO:4), ADQEANRHGRSGKDPNYYRPPGLPDKY (SEQ ID NO:8),ADQEANRHGRSGKDPNYYRPPGLPAKY (D-form; SEQ ID NO:10), ADQEANRHGRSGKDPNYYR(SEQ ID NO:25), ADQAANKWGRSGRDPNHFR (SEQ ID NO:11), ADQAANEWGRSGKDPNHFR(SEQ ID NO:12), or DQAANKWGRSGRDPNHFR (SEQ ID NO:26), or a peptidevariant of one of these peptides or a portion thereof or a peptidevariant of ADQEANRHGRSGKDPNYYRPPGLPAKY (SEQ ID NO:9) orADQAANEWGRSGKDPNHFRPAGLPEKY (SEQ ID NO:24). Excluded from the scope ofthe peptides of the present invention capable of enhancing cholesterolester hydrolase activity are those isolated peptides consistingGFFSFIGEAFQGAGDMWRAYTDMKEAGWKDGDKYFHARGNYDAAQRGPGGVWAAEKISD ARESFQEFFGRGHEDTMADQEANRHGRSGKDPNYYRPPGLPAKY (full length murine SAA1.1; SEQID NO:18); GFFSFVHEAFQGAGDMWRAYTDMKEANWKNSDKYFHARGNYDAAQRGPGGVWAAEKISDGREAFQEFFGRGHEDTIADQEANRHGRSGKDPNYYRPPGLPDKY (full length murine SAA2.1;SEQ ID NO:19);RSFFSFLGEAFDGARDMWRAYSDMREANYIGSDKYFHARGNYDAAKRGPGGVWAAEAISDARENIQRFFGHGAEDSLADQAANEWGRSGKDPNHFRPAGLPEKY (full length human SAA1.1;SEQ ID NO:20);RSFFSFLGEAFDGARDMWRAYSDMREANYIGSDKYFHARGNYDAAKRGPGGAWAAEVISNARENIQRLTGHGAEDSLADQAANKWGRSGRDPNHFRPAGLPEKY (full length human SAA2.1;SEQ ID NO:21);KEAGWKDGDKYFHARGNYDAAQRGPGGVWAAEKISDARESFQEFFGRGHEDTMADQEANRHGRSGKDPNYYRPPGLPAKY (SEQ ID NO:22);KEANWKNSDKYFHARGNYDAAQRGPGGVWAAEKISDGREAFQEFFGRGHEDTIADQEANRHGRSGKDPNYYRPPGLPDKY (SEQ ID NO:23); ADQEANRHGRSGKDPNYYRPPGLPAKY (SEQID NO:9); or ADQAANEWGRSGKDPNHFRPAGLPEKY (SEQ ID NO:24).

Also preferred for use in the present invention to enhance cholesterolester hydrolase activity and/or inhibit acyl CoA:cholesterol acyltransferase activity are compounds with a formula of Y—Z or Q-Y—Z. Inthese compounds Z is linked to Y and/or Q is linked to Y—Z via anyacceptable binding means and selected based upon selection of Z or Q.Examples of acceptable binding means include, but are in no way limitedto, covalent binding, noncovalent binding, hydrogen binding,antibody-antigen recognition, or ligand binding. In compounds with theformula Y—Z or Q-Y—Z, Y comprises an isolated peptide or mimetic of thepresent invention with cholesterol ester hydrolase enhancing activityand/or acyl CoA:cholesterol acyl transferase inhibitory activity; Zcomprises a compound linked to Y that enhances the performance of Y; andin embodiments comprising Q, Q may be identical to Z or different from Zand also enhances performance of the compound Q-Y—Z. Exemplary Z or Qcompounds include, but are not limited to, a targeting agent a secondagent for treatment of atherosclerosis, cardiovascular disease orcoronary heart disease, an agent which enhances solubility, absorption,distribution, half-life, bioavailability, stability, activity and/orefficacy, or an agent which reduces toxicity or side effects of thecompound. Exemplary targeting agents of Z and/or Q include macrophagetargeting agents such as, for example, a liposome, a microsphere, or aligand for a SAA receptor, hepatic targeting agents, antibodies andactive fragments thereof such as, for example, Fab fragments, andadditional agents specific to atherosclerotic plaques and/orinflammatory sites.

By “isolated” as used herein it is meant a peptide substantiallyseparated from other cellular components that naturally accompany thenative peptide or protein in its natural host cell. The term is meant tobe inclusive of a peptide that has been removed from its naturallyoccurring environment, is not associated with all or a portion of apeptide or protein in which the “isolated peptide” is found in nature,is operatively linked to a peptide to which it is not linked or linkedin a different manner in nature, does not occur in nature as part of alarger sequence or includes amino acids that are not found in nature.The term “isolated” also can be used in reference to recombinantlyexpressed peptides, chemically synthesized peptides, or peptide analogsthat are biologically synthesized by heterologous systems.

By “human equivalent” as used herein, it is meant a peptide sequencederived from human SAA2.1 or human SAA1.1 with similar activity to thereferenced murine peptides herein.

By the phrase “derived from” it is meant to include peptides or mimeticsthat originated from a particular species and were isolated from thatparticular species as well as peptides identical in amino acid sequencewhich are recombinantly expressed in a host cell expression system orchemically synthesized.

By “mimetic” as used herein it is meant to be inclusive of peptides,which may be recombinant, and peptidomimetics, as well as small organicmolecules, which exhibit similar or enhanced acyl CoA:cholesterol acyltransferase, and/or cholesterol ester hydrolase modulating activity.These include peptide variants which comprise conservative amino acidsubstitutions relative to the sequence of the native domains of SAA2.1or SAA1.1 and peptide variants which have a high percentage of sequenceidentity with the native domains of SAA2.1 or SAA1.1, at least e.g. 80%,85%, 90%, preferably at least 95%, 96%, 97%, 98%, or 99% sequenceidentity, and more preferably at least 99.5% or 99.9% sequence identity.Variant peptides can be aligned with the reference peptide to assesspercentage sequence identity in accordance with any of the well-knowntechniques for alignment. For example, a variant peptide greater inlength than a reference peptide is aligned with the reference peptideusing any well known technique for alignment and percentage sequenceidentity is calculated over the length of the reference peptide,notwithstanding any additional amino acids of the variant peptide, whichmay extend beyond the length of the reference peptide.

Preferred variants include, but are not limited to, peptides comprisingone or more D amino acids, which are equally effective but lesssusceptible to degradation in vivo, and cyclic peptides. Cyclic peptidescan be circularized by various means including but not limited topeptide bonds or depsicyclic terminal residues (i.e. a disulfide bond).

Also preferred is a variant comprising two or more linked or conjugatedpeptides of the present invention. Particularly preferred is a variantcomprising a peptide capable of enhancing cholesterol ester hydrolaseactivity linked or conjugated to a peptide capable of inhibiting acylCoA:cholesterol acyl transferase activity.

As used herein, the term “peptidomimetic” is intended to include peptideanalogs that serve as appropriate substitutes for the peptides of SEQ IDNO:1, 4, 6, 7, 8, 9, 10, 11, 12, 13, 14, 24, 25, 26 or 29 in modulatingacyl CoA:cholesterol acyl transferase and/or cholesterol ester hydrolaseactivity. The peptidomimetic must possess not only similar chemicalproperties, e.g. affinity, to these peptide domains, but also efficacyand function. That is, a peptidomimetic exhibits function(s) of an acylCoA:cholesterol acyl transferase inhibitory domain of SAA2.1 and/or acholesterol ester hydrolase enhancing domain of SAA2.1, withoutrestriction of structure. Peptidomimetics of the present invention, i.e.analogs of the acyl CoA:cholesterol acyl transferase inhibitory domainof SAA2.1 and/or the cholesterol ester hydrolase enhancing domain ofSAA2.1, include amino acid residues or other moieties which provide thefunctional characteristics described herein. Peptidomimetics and methodsfor their preparation and use are described in Morgan et al. 1989,“Approaches to the discovery of non-peptide ligands for peptidereceptors and peptidases,” In Annual Reports in Medicinal Chemistry(Vuirick, F. J. ed), Academic Press, San Diego, Calif., 243-253.

Mimetics of the present invention may be designed to have a similarstructural shape to the acyl CoA:cholesterol acyl transferase inhibitorydomain of SAA2.1 or the cholesterol ester hydrolase enhancing domain ofSAA2.1. For example, mimetics of the acyl CoA:cholesterol acyltransferase inhibitory domain of SAA2.1 of the present invention can bedesigned to include a structure which mimics aromatic amino acids suchas those characterized by (X)_(n)FFX₁FX₂X₃X₄X₅FX₆ (SEQ ID NO:13 or SEQID NO:14), e.g. residues 1-11 of SEQ ID NO:1, residues 2-12 of SEQ IDNO;6 or residue 1-12 of SEQ ID NO:7, and which is folded or stacked(e.g. pi-bonded) in an appropriate conformation to exhibit activity ofinhibition of acyl CoA:cholesterol acyl transferase. The efficacy ofmimetics of the present invention having aromatic regions as acylCoA:cholesterol acyl transferase inhibitors is also reasonably expectedin light of the aromaticity found in various known ACAT inhibitors(McCarthy et al. J. Med. Chem. 1994 37:1252-1255). For polypeptidemimetics or peptidomimetics of the present invention mimicking thestacked or folded aromatic amino acids of the acyl CoA:cholesterol acyltransferase inhibitory domain of SAA2.1, preferred amino acids forinclusion include, but are not limited to, trytophan, phenylalanine,histidine and tyrosine.

Mimetics of the present invention with cholesterol ester hydrolaseenhancing domain may also be designed to include a structure whichmimics the salt bridge conformation ofX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁X₁₂X₁₃X₁₄X₁₅X₁₆X₁₇X₁₈ (SEQ ID NO:29) or aportion thereof wherein X₁ and X₉, X₁₂ or X₁₈ are amino acids capable offorming a salt bridge, X₆ is glutamic acid or lysine or an amino acidwhich is a conservative substitution thereof, and X₂, X₃, X₄, X₅, X₇,X₈, X₁₀, X₁₁, X₁₃, X₁₄, X₁₅, X₁₆, and X₁₇ are independently any aminoacid.

Mimetics of the acyl CoA:cholesterol acyl transferase inhibitory domainof SAA2.1 or the cholesterol ester hydrolase enhancing domain of SAA2.1can also be designed to have a similar structure to the syntheticpeptides of SEQ ID NO:1, 6, 7, 13 or 14, or SEQ ID NO:4, 8, 9, 10, 11,12, 24, 25, 26 or 29, respectively. These, peptidomimetics may comprisepeptide sequences with conservative amino acid substitutions as comparedto SEQ ID NO:1, 6, 7, 13 or 14 or SEQ ID NO:4, 8, 9, 10, 11, 12, 24, 25,26 or 29 which interact with surrounding amino acids to form a similarstructure to these synthetic peptides. Conformationally restrictedmoieties such as a tetrahydroisoquinoline moiety may also be substitutedfor a phenylalanine, while histidine bioisoteres may be substituted forhistidine to decrease first pass clearance by biliary excretion.Peptidomimetics of the present invention may also comprise peptidebackbone modifications. Analogues containing amide bond surrogates arefrequently used to study aspects of peptide structure and functionincluding, but not limited to, rotational freedom in the backbone,intra- and intermolecular hydrogen bond patterns, modifications to localand total polarity and hydrophobicity, and oral bioavailability.Examples of isosteric amide bond mimics include, but are not limited to,ψ[CH₂S], ψ[CH₂NH], ψ[CSNH₂], ψ[NHCO], ψ[COCH₂] and ψ[(E) or (Z)CH═CH].

Mimetics can also be designed with extended and/or additional amino acidresidue repeats as compared to the naturally occurring acylCoA:cholesterol acyl transferase inhibitory domain of SAA2.1 and/or thecholesterol ester hydrolase enhancing domain of SAA2.1. For example,mimetics comprising two or more repeats of (X)_(n)FFX₁FX₂X₃X₄X₅FX₆ (SEQID NO:13 or SEQ ID NO:14), e.g. residues 1-11 of SEQ ID NO:1, residues2-12 of SEQ ID NO;6 or residue 1-12 of SEQ ID NO:7, portion of the acylCoA:cholesterol acyl transferase inhibitory domain, which may be flankedand/or separated by stabilizing amino acids, may be active inhibitors ofacyl CoA:cholesterol acyl transferase. Alternatively, such repeats maycontain one or more substitutions of one aromatic amino acid for anotheraromatic amino acid, e.g. W, H, or Y for F. Further, amino acids ofthese peptides believed to be important to the activity and/or stabilityof the conformation of the peptides, such as the initial arginine of SEQID NO:6 which is believed to form a hydrogen bond with an internalresidue in the region of residues 12-13 of SEQ ID NO:6, may beincorporated into mimetics to enhance their activity and/or stability.Host cells can be genetically engineered to express such mimetics inaccordance with routine procedures.

Identification of these peptide domains also permits molecular modelingbased on these peptides for design, and subsequent synthesis, of smallorganic molecules that have cholesterol ester hydrolase enhancing and/oracyl CoA:cholesterol acyl transferase-inhibiting activities. These smallorganic molecules mimic the structure and activity of the peptides ofSEQ ID NO:1, 4, 6, 7, 8, 9, 10, 11, 12, 13, 14, 24, 25, 26 or 29.However, instead of comprising amino acids, these small organicmolecules comprise bioisosteres thereof, substituents or groups thathave chemical or physical similarities, and exhibit broadly similarbiological activities.

Bioisosterism is a lead modification approach used by those skilled inthe art of drug design and shown to be useful in attenuating toxicityand modifying activity of a lead compound such as SEQ ID NO:1, 4, 6, 7,8, 9, 10, 11, 12, 13, 14, 24, 25, 26 or 29. Bioisosteric approaches arediscussed in detail in standard reference texts such as The OrganicChemistry of Drug Design and Drug Action (Silverman, R B, AcademicPress, Inc. 1992 San Diego, Calif., pages 19-23). Classical bioisosterescomprise chemical groups with the same number of valence electrons butwhich may have a different number of atoms. Thus, for example, classicalbioisosteres with univalent atoms and groups include, but are notlimited to: CH₃, NH₂, OH, F and Cl; C₁, PH₂ and SH; Br and i-Pr; and Iand t-Bu. Classical bioisosteres with bivalent atoms and groups include,but are not limited to: —CH₂— and NH; O, S, and Se; and COCH₂, CONHR,CO₂R and COSR. Classical bioisosteres with trivalent atoms and groupsinclude, but are not limited to: CH═ and N═; and P═ and As═. Classicalbioisosteres with tetravalent atoms include, but are not limited to: Cand Si; and ═C⁺═, ═N⁺═ and ═P⁺═. Classical bioisosteres with ringequivalents include, but are not limited to: benzene and thiophene;benzene and pyridine; and tetrahydrofuran, tetrahydrothiophene,cyclopentane and pyrrolidine. Nonclassical bioisosteres still produce asimilar biological activity, but do not have the same number of atomsand do not fit the electronic and steric rules of classical isosteres.Exemplary nonclassical bioisoteres are shown in the following Table.

Nonclassical Biosteres 1. Carbonyl group

2. Carboxylic acid group

3. Hydroxy group

—NHSO₂R —CH₂OH

—NHCN —CH(CN)₂ 4. Catachol

5. Halogen

CN CF₃ N(CN)₂ C(CN)₃ 6. Thioether

7. Thiourea

8. Azomethine

9. Pyridine

10. Spacer group

11. Hydrogen

FAdditional bioisosteric interchanges useful in the design of smallorganic molecule mimetics of the present invention include ring-chaintransformations.

A peptide or portion thereof, Y—Z or Q-Y—Z compound or mimetic thereofof the present invention is preferably formulated with a vehiclepharmaceutically acceptable for administration to a subject, preferablya human, in need thereof. Methods of formulation for such compositionsare well known in the art and taught in standard reference texts such asRemington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.,1985. A composition of the present invention may comprise a singlepeptide or portion thereof, a Y—Z or Q-Y—Z compound, or a mimetic ofthese which modulates either acyl CoA:cholesterol acyl transferaseactivity or cholesterol ester hydrolase activity, or both acylCoA:cholesterol acyl transferase activity and cholesterol esterhydrolase activity. Further, compositions of the present invention maycomprise a peptide of SEQ ID NO: 1, 6, 7, 13 or 14 or a portion or amimetic thereof and a peptide of SEQ ID NO:4, 8, 9, 10, 11, 12, 24, 25,26 or 29 or a portion or a mimetic thereof. These compositions may beadministered alone or in combination with a second cholesterol-loweringdrug or agent. For example, a composition of the present inventioncomprising a peptide of SEQ ID NO:4, 8, 9, 10, 11, 12, 24, 25, 26 or 29or a mimetic thereof which inhibits cholesterol ester hydrolaseactivity, can be administered to a subject in combination with an ACATinhibitor. Exemplary ACAT inhibitors include but are not limited toZetia™ (Merck), Avasimibe (Pfizer), Eflucimibe (Eli Lilly) and CS-505(Sankyo). Compositions of the present invention may also be administeredto a subject with an apolipoprotein-free acceptor such as cyclodextrin.Additional exemplary cholesterol-lowering drugs or agents which can beadministered in combination with an isolated peptide or mimetic of thepresent invention include, but are not limited to, statins, resins orbile acid sequestrants (Bays et al. Expert Opinion on Pharmacotherapy2003 4(11):1901-38; Kajinami et al. Expert Opinion on InvestigationalDrugs 2001 11(6):831-5), niacin (Van et al. Am. J. Cardiol. 200289(11):1306-8; Ganji et al. J. Nutri. Biochem. 2003 14(6):298-305;Robinson et al. Progress in Cardiovasc. Nursing 2001 16(1):14-20; Knopp,R. H. Am. J. Cardiol. 2000 86(12A):51L-56L), liver X receptor agonists(Tontonoz et al. Molecular Endocrinology 2003 17:985-993), Ca2+antagonists (Delsing et al. Cardiovasc. Pharmacol. 2003 42(1):63-70) andmodulators of peroxisome proliferator-activated receptors (PPARs; Lee etal. Endocrinology 2003 144:2201-2207).

A preferred formulation for use in the present invention is complexingthe peptide or mimetic thereof or Y—Z or Q-Y—Z compound with a lipid.Also preferred as a formulation is encapsulation of the peptide ormimetic thereof or Y—Z or Q-Y—Z compound or mimetic thereof in aphospholipid vesicle. As demonstrated throughout the instantapplication, an exemplary phospholipid vesicle useful in the presentinvention is a liposome. Liposomes containing the peptide or mimeticthereof or Y—Z or Q-Y—Z compound or mimetic thereof of the presentinvention can be prepared in accordance with any of the well knownmethods such as described by Epstein et al. (Proc. Natl. Acad. Sci. USA82: 3688-3692 (1985)), Hwang et al. (Proc. Natl. Acad. Sci. USA 77:4030-4034 (1980)), EP 52,322, EP 36,676; EP 88,046; EP 143,949; EP142,641; Japanese Pat. Appl. 83-118008, and EP 102,324, as well as U.S.Pat. Nos. 4,485,045 and 4,544,545, the contents of which are herebyincorporated by reference in their entirety. Preferred liposomes are ofthe small (about 200-800 Angstroms) unilamellar type in which the lipidcontent is greater than about 10 mol. percent cholesterol, preferably ina range of 10 to 40 mol. percent cholesterol, the selected proportionbeing adjusted for optimal peptide therapy. However, as will beunderstood by those of skill in the art upon reading this disclosure,phospholipid vesicles other than liposomes can also be used.

The peptides, compounds and mimetics of these or pharmaceuticalcompositions of the present invention can also be administered via acoronary stent implanted into a patient. Coronary stents which elute apeptide, compound and mimetic of these or a pharmaceutical compositionof the present invention can be prepared and implanted in accordancewith well known techniques (See, for example, Woods et al. (2004) Annu.Rev. Med. 55:169-78); al-Lamce et al. (2003) Med. Device Technol. 200314:12-141 Lewis et al. 2002 J. Long Term Eff. Med. Implants 12:231-50;Tsuji et al. 2003 Int. J. Cardiovasc. Intervent. 5:13-6).

Pharmaceutical compositions of the present invention are useful inmodifying the activity of a cholesterol-metabolizing enzyme, and inparticular, the activity of cholesterol ester hydrolase and/or acylCoA:cholesterol acyl transferase. In a preferred embodiment, thepharmaceutical compositions are used to modify enzymatic activity inmacrophages. More preferably, the pharmaceutical compositions are usedto modify enzymatic activity in vivo. More preferably, thepharmaceutical compositions are used to modify enzymatic activity inmammals and in particular humans.

Pharmaceutical compositions of the present invention are also useful inpromoting the mobilization and efflux of stored cholesterol located inatherosclerotic plaques and/or sites of inflammation. In a preferredembodiment, the pharmaceutical compositions are used to promote themobilization and efflux of stored cholesterol from macrophages and othertissues located in atherosclerotic plaques or sites of inflammation invivo. More preferably, the pharmaceutical compositions are used topromoting the mobilization and efflux of stored cholesterol frommacrophages and other tissues located in atherosclerotic plaques orsites of inflammation in mammals and in particular humans.

Accordingly, the compositions of the present invention can beadministered to a subject, preferably a mammal, more preferably a human,to treat and/or prevent atherosclerosis. The compositions may beadministered by various routes including, but not limited to, orally,intravenously, intramuscularly, intraperitoneally, topically, rectally,dermally, sublingually, buccally, intranasally or via inhalation. For atleast oral administration, it may be preferred to administer acomposition comprising a peptide with one or more D amino acids. Theformulation and route of administration as well as the dose andfrequency of administration can be selected routinely by those skilledin the art based upon the severity of the condition being treated, aswell as patient-specific factors such as age, weight and the like. Theprolonged activity of synthetic peptides of the present invention inpromoting cholesterol efflux from macrophages is indicative of thefeasibility of daily, every other day or semi-weekly dosing regime forthese pharmaceutical compositions.

In addition to the above-described in vitro and in vivo assays, efficacyof compositions of the present invention to treat and/or preventatherosclerosis can also be demonstrated in an animal model such as theApoE knockout mouse model of atherogenesis (Davis et al. ArteriosclerThromb Vasc Biol. 2001 21:2031-2038). These mice, when placed on anatherogenic diet, rapidly deposit lipid into their aortas. The ApoEknockout mice are a validated model of atherosclerosis and were used todemonstrate the effectiveness of Ezetimibe (Zetia™; Merck) in reducingatherosclerosis (Davis et al. Arterioscler Thromb Vasc Biol. 200121:2031-2038). The efficacy of compositions of the present invention,such as, e.g., those comprising one or more peptides of SEQ ID NO: 1, 4or 6 or a mimetic thereof, in treating or preventing atherosclerosis canbe demonstrated in similar fashion.

The in vivo effectiveness of a composition of the present invention,such as a composition comprising a peptide of SEQ ID NO:1, 4, 6, 7, 8,9, 10, 11, 12, 13, 14, 24, 25, 26 or 29 in preventing or reducing thedegree of atherosclerosis, can be demonstrated in the above rodent modelfor atherogenesis. To demonstrate the ability of a composition of thepresent invention to cause regression of atherosclerosis, the rodent isplaced on an atherogenic diet, such as described in Example 11, for twoweeks. The animals are then divided into two groups, one group whichcontinues on the diet for an additional two weeks, the other group whichcontinues on the diet for the same period but also receives acomposition of the present invention. The effects of a composition ofthe present invention on aortic atherosclerosis are assessed at thetermination of the experiment, when the aorta is removed from theanimals and opened longitudinally. The area of the endothelial surfaceoccupied by lipid is measured. Histological sections of aorta are alsoprepared for microscopic analysis and total lipids are isolated tomeasure the quantity of cholesterol per wet weight of tissue.

This rodent model was used to examine the anti-atherogenic activities ofSAA2.1 peptides (SEQ ID NO: 1 and a combination thereof) in vivo. Liversfrom SAA2.1 peptide-treated mice exhibited a more normal reddish colorin comparison to the whitish color observed in fatty livers of untreatedmice. These data are indicative of these SAA2.1 peptides modulatingcholesterol metabolism within the liver, as well as modulatingmacrophage cholesterol metabolism.

Further, the ability of liposomal formulations containing peptides ofthe present invention to prevent and induce regression of aortic lesionsin the ApoE knockout mice was examined.

In regression experiments, ApoE knockout mice were placed on anatherogenic diet as described in Example 11 for four weeks, followingwhich they were divided into two groups. One group continued on the dietfor an additional two weeks. The other group continued on the diet forthe same period but also received once every four days liposomescontaining a peptide corresponding to amino acids 1-20 of murine SAA2.1(SEQ ID NO:1; Group B of FIG. 7A and Group hf+p1 of FIG. 7B) orliposomes containing a peptide corresponding to amino acids 74-103 ofmurine SAA2.1 (SEQ ID NO:4; Group D of FIG. 7A and Group hf+p4 of FIG.7B). The control group received high fat diet alone with no liposomes(Group A of FIG. 7A and Group high fat of FIG. 7B). An additional groupwas placed on a normal mouse chow diet (Group C of FIG. 7A and Group lowfat of FIG. 7B). A further additional Group receiving liposomescontaining a peptide corresponding to amino acids 1-20 of murine SAA2.1(SEQ ID NO:1) and liposomes containing a peptide corresponding to aminoacids 74-103 of murine SAA2.1 (SEQ ID NO:4) is depicted in FIG. 7B andis referred to as hf+p1+p4. FIG. 7B is inclusive of data present in FIG.7A as well as data from a subsequent experiment performed under the sameconditions. After the two weeks, the mice were killed and their aortaswere dissected and stained with Oil Red O. Data of FIG. 7A depict thearea stained with Oil Red O indicative of the actual lipid positive areaas a percentage of the total aortic area viewed. Data of FIG. 7B depictthe area stained with Oil Red O as a percentage relative to the high fatdiet group (100%). As shown in these Figures, mice treated with aliposomal formulation containing a peptide of the present inventionshowed regression of aortic lesions as compared to control animals onthe high fat diet.

In prevention experiments, ApoE knockout mice were placed on a high fatdiet and at the same time received once every four days liposomescontaining a peptide corresponding to amino acids 1-20 of murine SAA2.1(SEQ ID NO:1; Group 2 of FIG. 8A and Group hf+p1 of FIG. 8B), liposomescontaining a peptide corresponding to amino acids 74-103 of murineSAA2.1 (SEQ ID NO:4; Group 4 of FIG. 8A and Group hf+p4 of FIG. 8B) orliposomes containing a peptide corresponding to amino acids 1-20 ofmurine SAA2.1 and a peptide corresponding to amino acids 74-103 ofmurine SAA2.1 (SEQ ID NO:1+SEQ ID NO:4; Group 5 of FIG. 8A and Grouphf+p(1+4) of FIG. 8B). The control group received high fat diet alonewith no liposomes (Group 1 of FIG. 8A and Group high fat of FIG. 8B). Anadditional group was placed on a normal mouse chow diet (Group 3 of FIG.8A and Group low fat of FIG. 8B). In FIG. 8B an additional experimentalgroup referred to as “empty lipos” was included which are animals thatwere treated with empty liposomes identical to the peptide containingliposomes but which are protein-peptide free. This group is differentfrom the high fat and low fat (diet) groups that were not treated withliposomes. Data present in FIG. 8B is inclusive of data presented inFIG. 8A and a subsequent experiment performed under the same conditions.After 20 days, the mice were killed and their aortas were dissected andstained with Oil Red O. Data from these experiments are depicted in FIG.8A and FIG. 8B. As shown therein, mice treated with a liposomalformulation containing a peptide of the present invention showeddecreased aortic lesions as compared to the control animals.

These experiments in this well-accepted rodent model of atherosclerosisprovide further evidence of pharmaceutical compositions of the presentinvention comprising an SAA peptide or mimetic modulating cholesterolmetabolic pathways in various tissues and/or cells. Using techniquessuch as pharmacokinetic scaling, these studies in rodents can be used topredict disposition and define pharmacokinetic equivalence and to designdosage regimens in other species including humans (Mordenti, J. (1986)J. Pharmaceutical Sciences 75(11):1028-1040).

Administration of pharmaceutical compositions of the present inventionis also expected to be useful in the treatment of coronary heart diseaseand cardiovascular disease and in the prevention or treatment ofinflammation.

The invention is further illustrated by the following examples, whichshould not be construed as further limiting. The contents of allreferences, pending patent applications, and published patents citedthroughout this application are hereby expressly incorporated byreference.

EXAMPLES Example 1 Animals

Swiss-white CD1 6-8 week old female mice were obtained from CharlesRiver, Montreal, Quebec. Mice were kept in a temperature controlled roomon a 12 hour light/dark cycle. They were fed with Purina Lab Chowpellets and water ad libitum.

ApoE knockout mice were obtained from Jackson Laboratories, Maine,U.S.A.

Example 2 Chemicals

All chemicals were reagent grade and purchased from Fisher Scientific(Nepean, Ont.), Sigma (St. Louis, Mo.), ICN (Aurora, Ohio), or BioRad(Hercules, Calif.). Dulbecco's Modified Eagle Medium (DMEM) and fetalbovine serum (FBS) were purchased from Life Technologies (Burlington,Ont.). Radiolabeled [1-¹⁴C]-oleic acid (52 mCi/mmol),[1,2,6,7-³H(N)]-cholesterol (82 Ci/mmol), andcholesteryl-1,2,6,7-³H(N)]-oleate (84 Ci/mmol) were obtained from DuPontNEN (Boston, Mass.).

Example 3 Peptides

The following peptides were synthesized by solid-phase peptide synthesisusing 9-fluorenylmethoxycarbonyl as an ∀-amino protecting group in a PEApplied Biosystems 433A peptide synthesizer:

GFFSFVHEAFQGAGDMWRAY  (SEQ ID NO: 1) TDMKEANWKNSDKYFHARGNYDAAQRGPGG(SEQ ID NO: 2) VWAAEKISDGREAFQEFFGRGHEDTIADQE (SEQ ID NO: 3)DTIADQEANRHGRSGKDPNYYRPPGLPDKY (SEQ ID NO: 4) GFFSFIGEAFQGAGDMWRAY(SEQ ID NO: 5) RSFFSFLGEAFDGARDMWRAYSD (SEQ ID NO: 6)RGFFSFIGEAFQGAGDMWRAY (SEQ ID NO: 7) ADQEANRHGRSGKDPNYYRPPGLPDKY(SEQ ID NO: 8) ADQEANRHGRSGKDPNYYRPPGLPAKY (SEQ ID NO: 9)ADQAANKWGRSGRDPNHFR (SEQ ID NO: 11) ADQAANEWGRSGKDPNHFR (SEQ ID NO: 12)The purity of the synthetic peptides was established by analytical highperformance liquid chromatography (HPLC) and ion-spray massspectrometry. The peptides were dialyzed against distilled water andlyophilized before use.

Example 4 Preparation of Red Blood Cell Membranes as a Source ofCholesterol

To mimic the ingestion of cell membrane fragments by macrophages atsites of tissue injury, red blood cell membrane fragments were preparedand used as a source of cholesterol in accordance with the proceduredescribed by Ely et al. (Amyloid 2001 8:169-181). Similar quantities ofcholesterol (as red blood cell membrane fragments) were used in allexperiments. The concentration of cholesterol in the red blood cellmembrane preparations was determined using the method of Allain andco-workers (Clin. Chem. 1974 20:470-475), with the aid of a Sigmacholesterol 20 reagent kit (Sigma Chemical Co., St. Louis, Mo.).

Example 5 Preparation of HDL, AP-HDL and Purification of apoA-1 and SAAIsoforms

HDL and AP-HDL were isolated from normal and inflamed mice,respectively, using sequential density flotation in accordance withprocedure described by Ancsin and Kisilevsky (Amyloid 1999 6:37-47; J.Biol. Chem. 1999 274:7172-7181). In this procedure, inflammation wasinduced by subcutaneous injection of 0.5 mL of 2% AgNO₃ under the looseskin of the upper back of the mice. Twenty-four hours later, after CO₂narcosis, the animals were exsanguinated by cardiac puncture and theblood collected into 0.5% EDTA (final concentration). The plasma wasthen separated from the red blood cells by centrifugation. The inductionof inflammation and SAA synthesis and the isolation of apoA-1, SAA1.1and 2.1 from acute phase murine plasma were performed as described byAncsin and Kisilevsky (J. Biol. Chem. 1999 274:7172-7181). Separationand purification of these proteins was accomplished by reverse phasehigh-pressure liquid chromatography as described by Ancsin andKisilevsky (Amyloid 1999 6:37-47). The purity of the isolated proteinswas established by mass spectrometry and N-terminal sequence analysis asdescribed by Ancsin and Kisilevsky (Amyloid 1999 6:37-47 and J. Biol.Chem. 1999 274:7172-7181).

Example 6 Preparation and Characterization of Apolipoprotein-LipidComplexes

ApoA-1, SAA1.1, SAA2.1, synthetic peptides corresponding to amino acidresidues 1-20 of murine SAA1.1 and 2.1, respectively, and syntheticpeptides corresponding to amino acid residues 21-50, 51-80 and 74-103 ofmurine SAA2.1 were reconstituted with lipids to form liposomes. Theseliposomes were made by the cholate dialysis procedure as described byJonas et al. (J. Biol. Chem. 1989 264:4818-4825), using1-palmitoyl-2-oleoylphosphatidylcholine/cholesterol/apolipoprotein/sodiumcholate in the molar ratio 100/25/1/250. Cholesterol was included tostabilize the liposomes and give them a composition more similar to thatof HDL. All preparations were done in 0.5 mL of buffer containing 10 mMTris-HCl, pH 7.4, 0.15 M NaCl and 0.005% EDTA. The reaction mixture wasstirred thoroughly and incubated for 12 to 16 hours at 4° C. At the endof the equilibration period the sample was dialyzed extensively inphosphate buffered saline at 4° C. After removing any un-reacted orprecipitated lipid by centrifugation at 15000×g, 15° C., for 1 hour, theliposomes were filtered on a 1.5×50 cm Sepharose CL-4B column. Followingconcentration, the liposomes were sterilized by filtration through a0.45 μm Millipore filter and mixed at various concentrations with tissueculture medium. The chemical compositions of various protein-containingliposomes were obtained from protein determinations using the method ofLowry et al. (J. Biol. Chem. 1951 193:265-275), phospholipiddeterminations using a colorimetric kit (Wako Chemicals USA, Richmond,Va.), and enzymatic analyses of free cholesterol (Sigma cholesterolreagent kit, Sigma Chemical Co. St. Louis, Mo.).

Example 7 Cell Culture

J774 macrophages (from American Type Culture Collection, Manassas, Va.;ATCC #T1B-67) were maintained at million cells per well and grown in 2mL of DMEM supplemented with 10% FBS to 90% confluence. The medium waschanged 3 times a week. In some experiments, nearly confluentmono-layers were cultured in the presence of chloroquine (100 :M) or8-bromo-cAMP (0.3 mM).

Example 8 Cholesterol Loading and Determination of Cell CholesterolEsterification

To load the cells with cholesterol, nearly confluent mono-layers werewashed 3 times with phosphate buffered saline containing 2 mg/mL fattyacid-free bovine serum albumin (PBS-BSA) and incubated for 5 hours inDMEM supplemented with 5% lipoprotein-depleted serum (LPDS) (d>1.25g/mL) and 175 μg of red blood cell membrane cholesterol. For the purposeof pool equilibration of added cholesterol, cell cultures were rinsedtwice with PBS-BSA and incubated overnight in DMEM containing 5% LPDS.The relative activity of acyl CoA:cholesterol acyl transferase wasdetermined in cholesterol-laden cells that had been cultured in mediumcontaining no liposomes, protein-free liposomes or liposomes containing0.5 :M synthetic peptides corresponding to amino acid residues 1-20 (SEQID NO:1), 21-50 (SEQ ID NO:2), 51-80 (SEQ ID NO:3) or 74-103 (SEQ IDNO:4) of murine SAA2.1, respectively. Following 3 hours incubation withthe above media, [¹⁴C]-oleate was added and the cells were incubated foranother 3 hour period (Mendez et al. J. Clin. Invest. 1994 94:1698-1705;Oram et al. Arterioscler. Thromb. 1991 11:403-414). Cells were chilledon ice and washed twice with PBS-BSA and twice with PBS. After additionof [³H]-cholesteryl oleate (6000 dpm/well) as an internal standard, thelipids were extracted from the labeled cells and analyzed by thin-layerchromatography as described by Mendez et al. (J. Clin. Invest. 199494:1698-1705) and Oram et al. (Arterioscler. Thromb. 1991 11:403-414).The radioactivity in appropriate spots was measured to determine theincorporation of radioactivity into cholesteryl esters as a measure ofacyl CoA:cholesterol acyl transferase activity.

Example 9 Rates of Hydrolysis of Cholesteryl Ester in J774 Cells

Newly confluent J774 cells were labeled with [¹⁴C]-oleate duringcholesterol loading with red blood cell membranes as described above.Cells were then incubated for up to 24 hours with 2 mL of DMEMcontaining 5% LPDS and 50 μg/mL of either native HDL, SAA-HDL, liposomescontaining 2 μmoles of apoA-1, SAA1.1 or 2.1, or liposomes containing0.5 :mol synthetic peptides corresponding to amino acid residues 1-20(SEQ ID NO:1), 21-50 (SEQ ID NO:2), 51-80 (SEQ ID NO:3) and 74-103 (SEQID NO:4) of murine SAA2.1. To determine the rate of cholesteryl esterhydrolysis, 2 μg/mL of the acyl CoA:cholesterol acyl transferaseinhibitor Sandoz 58-035 (propanimide,3-(decyldimethylsilyl)-N-[2-(4-methylphenyl)-1-phenylethyl]-(9Cl) wasadded during incubation with lipoproteins or liposomes to preventre-esterification of liberated [¹⁴C]-oleate and free cholesterol. Toexamine whether or not the rate of cholesteryl ester hydrolysis involvedthe lysosomal cholesteryl ester hydrolase, the cells were cultured inthe presence of 50 μg/mL of either native HDL and chloroquine or SAA-HDLand chloroquine (100 :M). Chloroquine is an agent that neutralizes thelysosomal proton gradient. At various time points, cellular lipids wereextracted and analyzed for cholesteryl ester radioactivity as describedabove.

Example 10 Cholesterol Efflux in Tissue Culture and In Vivo

J774 cells were loaded with cholesterol and incubated for 3 hours with0.5 μCi/mL [³H]-cholesterol, followed by an overnight equilibrationperiod. Cells were washed four times with PBS/BSA prior to the effluxstudies. Cells were then incubated at 37° C. with DMEM/BSA andcontaining 5% LPDS and 50 μg/mL of either native HDL, SAA-HDL, liposomescontaining 2 μmoles of apoA-1, SAA1.1 or 2.1, or liposomes containing0.5 :mol synthetic peptides corresponding to amino acid residues 1-20(SEQ ID NO:1), 21-50 (SEQ ID NO:2), 51-80 (SEQ ID NO:3) or 74-103 (SEQID NO:4) of murine SAA2.1 plus 2 μg/mL of the acyl CoA:cholesterol acyltransferase inhibitor Sandoz 58-035. The efflux media were collected at0, 1, 2, 4, 8, 16 and 24 hours, centrifuged to remove cell debris, andthen used to measure the exported counts. The cell layers were thenwashed twice with ice-cold PBS/BSA and twice with PBS. A portion of thecells was lysed in 0.1N NaOH to estimate both the remainingradioactivity and the cellular protein content. Cellular lipids wereextracted from the remaining portion of the cells and analyzed bythin-layer chromatography as described by Mendez et al. (J. Clin.Invest. 1994 94:1698-1705) and Oram et al. (Arterioscler. Thromb. 199111:403-414). The radioactivity in appropriate spots was measured todetermine total cellular cholesterol counts. Efflux of radioactive labelto the medium was calculated as the percentage of total counts(cell+medium counts) in each well.

To examine whether cholesterol export from J774 cells to mediumcontaining liposomes containing 2 μM murine apoA-1, SAA1.1 or SAA2.1 isa cAMP-dependent process, the radio-labeled cholesterol-laden cells wereincubated overnight with 8-Br-cAMP (0.3 mM), prior to the addition ofliposomes containing 2 μl murine apoA-1, SAA1.1 or SAA2.1 to the culturemedium. Cholesterol efflux to the medium was then determined at theindicated time points as described above. Efflux of radioactive label tothe medium was calculated as a percentage of total counts in each well.

To determine cholesterol export in vivo, J774 macrophages werecholesterol-loaded with red blood cell membranes and [³H]-cholesterol asdescribed above. Cells were washed four times with PBS/BSA and thendetached from the culture dishes. Five million cells in 200 μl DMEM wereinjected into control mice or inflamed mice through the tail vein. Atvarious time points, approximately 25 μl of blood were collected fromthe tail vein of each animal into heparinized capillary tubes and thencentrifuged for 5 minutes in an Adams Autocrit Centrifuge to separatered blood cells from plasma. Cholesterol efflux was determined bymeasuring the appearance of [³H]-cholesterol in plasma by scintillationspectrometry.

To study whether export of cholesterol from J774 cells to plasma ismediated by the ABCA1 transporter pathway, or due to the endogenousdestruction of the injected cells, radio-labeled cholesterol-laden cellswere incubated overnight with 400 μM (final concentration) of4,4′-diiso-thiocyanotostilbene-2,2′-disulfonic acid (DIDS), and washedfree of DIDS prior to their injection into un-inflamed and inflamedmice. Inflammation, in the form of a small sterile abscess, was inducedin the back by the subcutaneous injection of 0.5 mL of a 2% solution ofAgNO₃ as described by Kisilevsky et al. (Nat. Med. 1995 1:143-148).

Example 11 Assessing Efficacy of Peptides by Determination of Regressionof Atherosclerosis

The efficacy of SAA2.1 peptides in regression of atherosclerosis wasexamined. The peptides tested included SEQ ID NO:1 and SEQ ID NO:4. Thepeptides were injected intravenously once every four (4) days duringatherogenic induction for a period of 2 weeks (i.e. four doses at 6mg/kg).

To determine if these peptides cause regression of atherosclerosis, theanimals were placed on an atherogenic diet (Paigen's Atherogenic RodentDiet: Purina 5015 with cocoa butter, cholesterol and cholic acid(CI3002, Research Diets, Inc.)) for two weeks, following which they weredivided into two groups of 5 animals each. One group continued on thediet for an additional two weeks. The other group continued on the dietfor the same period but also received the liposome-containing peptides(4 doses, as described above).

To assess the effects of the peptides on aortic atherosclerosis, at thetermination of the experiment, the aorta was removed from the animalsand opened longitudinally. The endothelial surface was stained with OilRed O and the area occupied by lipid was measured by image analysis.Furthermore, histological sections of aorta were prepared formicroscopic analysis and total lipids were isolated to measure thequantity of cholesterol per wet weight of tissue. Blood was collected tomeasure total plasma cholesterol levels.

Livers from SAA2.1 peptide (SEQ ID NOs 1 and 4)-treated and untreatedmice were also collected. Total liver tissue cholesterol and LDL levelsare to be analyzed. Preliminary examination of the SAA2.1peptide-treated livers showed that they had a more normal reddish colorin comparison to the whitish color observed in livers of untreated mice.These data are the first to suggest that these SAA2.1 peptides modulatecholesterol metabolism within the liver, as well as modulatingmacrophage cholesterol metabolism. This further suggests that these SAApeptides may modulate cholesterol metabolic pathways in additionaltissues/cells.

Example 12 Assessing Efficacy of Peptides by Determination of Preventionof Atherosclerosis

The efficacy of SAA2.1 peptides in preventing atherosclerosis wasexamined. The peptides tested included SEQ ID NO:1 and SEQ ID NO:4 andan equimolar combination of both peptides. Liposomes containing thesepeptides were injected intravenously into 8-12 week old ApoE knockoutmice once every fours days during atherogenic induction for a period of2-3 weeks (five doses at 6 mg/kg for prevention experiments).

Animals were divided into 5 groups (5 animals per group). The negativecontrol group was placed on a normal chow diet, while the other fourgroups received an atherogenic diet (Paigen's Atherogenic Rodent diet,as described in Example 11). Among these groups, one group continued onthe high fat diet for three weeks. The other groups continued on thehigh fat diet for the same period but also received either liposomescontaining peptide SEQ ID NO: 1, liposomes containing peptide SEQ IDNO:4 or liposomes containing an equimolar combination of both of thesepeptides (5 doses as described in the preceding paragraph).

To assess the effects of the peptides on aortic atherosclerosis, at thetermination of the experiment, the aorta was removed from the animalsand opened longitudinally. The endothelial surface was stained with OilRed O and the area occupied by lipid was measured by image analysis.Furthermore, histological sections of aorta were prepared formicroscopic analysis and total lipids were isolated to measure thequantity of cholesterol per weight of tissue. Blood was isolated tomeasure total plasma cholesterol levels.

Example 13 Cholesterol Efflux in Tissue Culture Mediated by L-Amino Acidand D-Amino Acid Peptides Corresponding to Residues 77-103 of MurineSAA1.1

Macrophages loaded with [³H]cholesterol were pre-incubated in theabsence or presence of liposomes containing 0.5 :M cyanogenbromide-released peptides corresponding to amino acid residues 77-103 ofmurine SAA1.1 (SEQ ID NO:9), synthetic D-amino acid peptides of thecorresponding sequence (SEQ ID NO:10), or synthetic peptidescorresponding to the native L-amino acid residues 74-103 of murineSAA2.1 (SEQ ID NO:7). Following incubation, the cells were washedextensively with Dulbecco's modified Eagle medium (DMEM) containing 0.2%bovine serum albumin (BSA) to remove all radioactivity and liposomes inthe pre-incubation medium. The chase efflux media consisted of DMEM/BSAalone or medium containing HDL (50 μg/mL). See FIG. 2. The resultsrepresent cholesterol efflux to the acceptor, HDL, in the medium fromcells with various liposome pre-treatments. The efflux media werecollected as 1, 2, 4, 8, 16 and 24 hours and analyzed for [³H]cholesterol. Total [³H] cholesterol was (1.8-2.1)×10⁶ dpm/mg cellprotein.

Example 14 Cholesterol Efflux in Human Monocytic Cell Line, THP-1

Studies were carried out to determine whether murine SAA2.1 increasescholesterol export from a human derived monocytic cell line, THP-1(obtained from American Type Culture Collection, Manassas, Va.;ATCC#TIB-202). Human monocytes were cultured in T-75 flasks with 30 mlRPMI 1640 medium containing 2 mM L-glutamine, 4.5 g/L glucose, 10 mMHEPES, 1.0 mM sodium private and supplemented with 0.05 mM2-mercaptoethanol and 10% fetal bovine serum. Subsequently, five millioncells were placed in each well of a 6-well tissue culture plate. Themonocytes were differentiated into macrophages by treatment with phorbolmyristate acetate (100 nM). THP-1 macrophages were enriched withcholesterol by incubating with red blood cells membrane fragments (175μg as cholesterol) that had been previously labelled with 0.5 μCi/mL[³H]-cholesterol at 37° C. for 6 hours in 0.2% bovine serum albumin,followed by an overnight equilibration period. Cells were washed fourtimes with PBS/BSA prior to efflux studies. Cells were then incubated at37° C. with 2 mL RPMI-BSA containing 5% LPDS and 50 μg/mL of eithernative HDL, SAA-HDL, liposomes containing 2 μmoles of apoA-I, SAA1.1 or2.1, or liposomes containing 0.5 μmoles synthetic peptides correspondingto amino acid residues 1-20 (SEQ ID NO:1), 21-50 (SEQ ID NO:2), 51-80(SEQ ID NO:3) or 74-103 (SEQ ID NO:4) of murine SAA2.1. The efflux mediawere collected at 0, 1, 2, 4, 8, 16 and 24 hours, centrifuged to removecell debris, and then used to measure the exported counts. The celllayers were then washed twice with ice-cold PBS/BSA and twice with PBS.A portion of the cells was lysed in 0.1N NaOH to estimate both theremaining radioactivity and the cellular protein content. Cellularlipids were extracted from the remaining portion of the cells andanalyzed by thin-layer chromatography as described by Mendez et al. (J.Clin. Invest. 1994 94:1698-1705) and Oram et al. (Arterioscler. Thromb.1991 11:403-414). The radioactivity in appropriate spots was measured todetermine total cellular cholesterol counts. Efflux of radioactive labelto the medium was calculated as the percentage of total counts(cell+medium counts) in each well.

Example 15 Statistical Analysis

Unpaired Student's t tests were used to compare group means. A value ofP<0.05 was considered statistically significant. Histological sectionsof aorta were compared by ANOVA.

1. An isolated peptide or a mimetic thereof which enhances cholesterolester hydrolase activity, said isolated peptide comprising a formula:X₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁X₁₂X₁₃X₁₄X₁₅X₁₆X₁₇X₁₈ (SEQ ID NO:29) or aportion thereof wherein X₁ and X₉, X₁₂ or X₁₈ are amino acids capable offorming a salt bridge; X₆ is glutamic acid or lysine or an amino acidwhich is a conservative substitution thereof; and X₂, X₃, X₄, X₅, X₇,X₈, X₁₀, X₁₁, X₁₃, X₁₄, X₁₅, X₁₆, and X₁₇ are independently any aminoacid, wherein said isolated peptide or mimetic thereof has less than 80amino acid residues; and with the proviso that said isolated peptidedoes not consist of: (SEQ ID NO: 18)GFFSFIGEAFQGAGDMWRAYTDMKEAGWKDGDKYFHARGNYDAAQRGPGGVWAAEKISDARESFQEFFGRGHEDTMADQEANRHGRSGKDPNYYRPPGLP AKY; (SEQ ID NO: 19)GFFSFVHEAFQGAGDMWRAYTDMKEANWKNSDKYFHARGNYDAAQRGPGGVWAAEKISDGREAFQEFFGRGHEDTIADQEANRHGRSGKDPNYYRPPGLP DKY; (SEQ ID NO: 20)RSFFSFLGEAFDGARDMWRAYSDMREANYIGSDKYFHARGNYDAAKRGPGGVWAAEAISDARENIQRFFGHGAEDSLADQAANEWGRSGKDPNHFRPAGL PEKY; (SEQ ID NO: 21)RSFFSFLGEAFDGARDMWRAYSDMREANYIGSDKYFHARGNYDAAKRGPGGAWAAEVISNARENIQRLTGHGAEDSLADQAANKWGRSGRDPNHFRPAGL PEKY; (SEQ ID NO: 22)KEAGWKDGDKYFHARGNYDAAQRGPGGVWAAEKISDARESFQEFFGRGHEDTMADQEANRHGRSGKDPNYYRPPGLPAKY; (SEQ ID NO: 23)KEANWKNSDKYFHARGNYDAAQRGPGGVWAAEKISDGREAFQEFFGRGHEDTIADQEANRHGRSGKDPNYYRPPGLPDKY; (SEQ ID NO: 9)ADQEANRHGRSGKDPNYYRPPGLPAKY; or (SEQ ID NO: 24)ADQAANEWGRSGKDPNHFRPAGLPEKY.


2. The isolated peptide or a mimetic thereof of claim 1 wherein X₂ isglutamine or an amino acid which is a conservative substitution thereof;X₃ and X₄ are independently alanine or an amino acid which is aconservative substitution thereof; X₅ and X₁₅ are independentlyasparagine or an amino acid which is a conservative substitutionthereof; X₇ is tryptophan or an amino acid which is a conservativesubstitution thereof; X₈ and X₁₁ are independently glycine or an aminoacid which is a conservative substitution thereof; X₁₀ is serine or anamino acid which is a conservative substitution thereof; X₁₃ is asparticacid or an amino acid which is a conservative substitution thereof; X₁₄is proline or an amino acid which is a conservative substitutionthereof; X₁₆ is histidine or an amino acid which is a conservativesubstitution thereof; and/or X₁₇ is phenylalanine or an amino acid whichis a conservative substitution thereof.
 3. (canceled)
 4. The isolatedpeptide or mimetic thereof of claim 1 which has 18 to 79 amino acidresidues.
 5. The isolated peptide or mimetic thereof of claim 1comprising: (SEQ ID NO: 4) DTIADQEANRHGRSGKDPNYYRPPGLPDKY;(SEQ ID NO: 8) ADQEANRHGRSGKDPNYYRPPGLPDKY; (D-form; SEQ ID NO: 10)ADQEANRHGRSGKDPNYYRPPGLPAKY; (SEQ ID NO: 25) ADQEANRHGRSGKDPNYYR;(SEQ ID NO: 11) ADQAANKWGRSGRDPNHFR; (SEQ ID NO: 12)ADQAANEWGRSGKDPNHFR; or (SEQ ID NO: 26) DQAANKWGRSGRDPNHFR;

or a peptide variant of one of these peptides or a portion thereof. 6.The isolated peptide or mimetic thereof of claim 5 which has at least80% sequence identity with (SEQ ID NO: 4)DTIADQEANRHGRSGKDPNYYRPPGLPDKY; (SEQ ID NO: 8)ADQEANRHGRSGKDPNYYRPPGLPDKY; (SEQ ID NO: 9) ADQEANRHGRSGKDPNYYRPPGLPAKY;(D-form; SEQ ID NO: 10) ADQEANRHGRSGKDPNYYRPPGLPAKY; (SEQ ID NO: 24)ADQAANEWGRSGKDPNHFRPAGLPEKY; (SEQ ID NO: 25) ADQEANRHGRSGKDPNYYR;(SEQ ID NO: 11) ADQAANKWGRSGRDPNHFR; (SEQ ID NO: 12)ADQAANEWGRSGKDPNHFR; or (SEQ ID NO: 26) DQAANKWGRSGRDPNHFR;

or a portion thereof.
 7. The isolated peptide or mimetic thereof ofclaim 5 which has at least 90% sequence identity with (SEQ ID NO: 4)DTIADQEANRHGRSGKDPNYYRPPGLPDKY; (SEQ ID NO: 8)ADQEANRHGRSGKDPNYYRPPGLPDKY; (SEQ ID NO: 9) ADQEANRHGRSGKDPNYYRPPGLPAKY;(D-form; SEQ ID NO: 10) ADQEANRHGRSGKDPNYYRPPGLPAKY; (SEQ ID NO: 24)ADQAANEWGRSGKDPNHFRPAGLPEKY; (SEQ ID NO: 25) ADQEANRHGRSGKDPNYYR;(SEQ ID NO: 11) ADQAANKWGRSGRDPNHFR; (SEQ ID NO: 12)ADQAANEWGRSGKDPNHFR; or (SEQ ID NO: 26) DQAANKWGRSGRDPNHFR;

or a portion thereof.
 8. The isolated peptide or mimetic thereof ofclaim 5 which has at least 95% sequence identity with (SEQ ID NO: 4)DTIADQEANRHGRSGKDPNYYRPPGLPDKY; (SEQ ID NO: 8)ADQEANRHGRSGKDPNYYRPPGLPDKY; (SEQ ID NO: 9) ADQEANRHGRSGKDPNYYRPPGLPAKY;(D-form; SEQ ID NO: 10) ADQEANRHGRSGKDPNYYRPPGLPAKY; (SEQ ID NO: 24)ADQAANEWGRSGKDPNHFRPAGLPEKY; (SEQ ID NO: 25) ADQEANRHGRSGKDPNYYR;(SEQ ID NO: 11) ADQAANKWGRSGRDPNHFR; (SEQ ID NO: 12)ADQAANEWGRSGKDPNHFR; or (SEQ ID NO: 26) DQAANKWGRSGRDPNHFR;

or a portion thereof.
 9. The isolated peptide or mimetic thereof ofclaim 5 which has at least 99% sequence identity with (SEQ ID NO: 4)DTIADQEANRHGRSGKDPNYYRPPGLPDKY; (SEQ ID NO: 8)ADQEANRHGRSGKDPNYYRPPGLPDKY; (SEQ ID NO: 9) ADQEANRHGRSGKDPNYYRPPGLPAKY;(D-form; SEQ ID NO: 10) ADQEANRHGRSGKDPNYYRPPGLPAKY; (SEQ ID NO: 24)ADQAANEWGRSGKDPNHFRPAGLPEKY; (SEQ ID NO: 25) ADQEANRHGRSGKDPNYYR;(SEQ ID NO: 11) ADQAANKWGRSGRDPNHFR; (SEQ ID NO: 12)ADQAANEWGRSGKDPNHFR; or (SEQ ID NO: 26) DQAANKWGRSGRDPNHFR;

or a portion thereof.
 10. The isolated peptide or mimetic thereof ofclaim 5 which has one or more conservative amino acid substitutions inthe amino acid sequence of the peptide comprising: (SEQ ID NO: 4)DTIADQEANRHGRSGKDPNYYRPPGLPDKY; (SEQ ID NO: 8)ADQEANRHGRSGKDPNYYRPPGLPDKY; (SEQ ID NO: 9) ADQEANRHGRSGKDPNYYRPPGLPAKY;(D-form; SEQ ID NO: 10) ADQEANRHGRSGKDPNYYRPPGLPAKY; (SEQ ID NO: 24)ADQAANEWGRSGKDPNHFRPAGLPEKY; (SEQ ID NO: 25) ADQEANRHGRSGKDPNYYR;(SEQ ID NO: 11) ADQAANKWGRSGRDPNHFR; (SEQ ID NO: 12)ADQAANEWGRSGKDPNHFR; or (SEQ ID NO: 26) DQAANKWGRSGRDPNHFR;

or a portion thereof.
 11. The isolated peptide or mimetic thereof ofclaim 1 wherein the mimetic is a small organic molecule.
 12. Theisolated peptide or mimetic thereof of claim 1 which is preparedsynthetically or recombinantly.
 13. A compound having a formula:Y—Z wherein Y comprises a peptide or a mimetic thereof that enhancescholesterol ester hydrolase activity; and wherein Z comprises a compoundlinked to Y that enhances the performance of Y; with the proviso thatY—Z does not consist of (SEQ ID NO: 18)GFFSFIGEAFQGAGDMWRAYTDMKEAGWKDGDKYFHARGNYDAAQRGPGGVWAAEKISDARESFQEFFGRGHEDTMADQEANRHGRSGKDPNYYRPPGLP AKY; (SEQ ID NO: 19)GFFSFVHEAFQGAGDMWRAYTDMKEANWKNSDKYFHARGNYDAAQRGPGGVWAAEKISDGREAFQEFFGRGHEDTIADQEANRHGRSGKDPNYYRPPGLP DKY; (SEQ ID NO: 20)RSFFSFLGEAFDGARDMWRAYSDMREANYIGSDKYFHARGNYDAAKRGPGGVWAAEAISDARENIQRFFGHGAEDSLADQAANEWGRSGKDPNHFRPAGL PEKY; or(SEQ ID NO: 21) RSFFSFLGEAFDGARDMWRAYSDMREANYIGSDKYFHARGNYDAAKRGPGGAWAAEVISNARENIQRLTGHGAEDSLADQAANKWGRSGRDPNHFRPAGL PEKY.


14. The compound of claim 13 wherein Y comprises a cholesterol esterhydrolase enhancing peptide domain of a serum amyloid A protein.
 15. Thecompound of claim 13 wherein Y comprises a peptide or a mimetic thereofwhich enhances cholesterol ester hydrolase activity, said peptidecomprising a formula: (SEQ ID NO:  29)X₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁X₁₂X₁₃X₁₄X₁₅X₁₆X₁₇X₁₈

or a portion thereof wherein X₁ and X₉, X₁₂ or X₁₈ are amino acidscapable of forming a salt bridge; X₆ is glutamic acid or lysine or anamino acid which is a conservative substitution thereof; and X₂, X₃, X₄,X₅, X₇, X₈, X₁₀, X₁₁, X₁₃, X₁₄, X₁₅, X₁₆, and X₁₇ are independently anyamino acid.
 16. The compound of claim 13 wherein Z comprises a targetingagent, a second agent for treatment of atherosclerosis, cardiovasculardisease or coronary heart disease, an agent which enhances solubility,absorption, distribution, half-life, bioavailability, stability,activity and/or efficacy, or an agent which reduces toxicity or sideeffects of the compound.
 17. The compound of claim 13 further comprisingQ linked to Y—Z wherein Q is identical to Z or different from Z andwherein Q comprises a targeting agent, a second agent for treatment ofatherosclerosis, cardiovascular disease or coronary heart disease, anagent which enhances solubility, absorption, distribution, half-life,bioavailability, stability, activity and/or efficacy, or an agent whichreduces toxicity or side effects of the compound.
 18. A pharmaceuticalcomposition comprising the isolated peptide or mimetic thereof of claim1 and a pharmaceutically acceptable vehicle.
 19. The pharmaceuticalcomposition of claim 18 further comprising a second agent for treatmentof atherosclerosis, cardiovascular disease or coronary heart disease.20. The pharmaceutical composition of claim 18 wherein the isolatedpeptide or mimetic thereof is complexed with a lipid.
 21. Thepharmaceutical composition of claim 18 wherein the isolated peptide ormimetic thereof is enclosed in a phospholipid vesicle. 22-78. (canceled)79. A pharmaceutical composition comprising an isolated peptide or amimetic thereof which enhances cholesterol ester hydrolase activity andan isolated peptide or a mimetic thereof which inhibits acylCoA:cholesterol acyl transferase.
 80. The pharmaceutical composition ofclaim 79 wherein the isolated peptide or mimetic thereof which enhancescholesterol ester hydrolase activity is linked to the isolated peptideor mimetic thereof which inhibits acyl CoA:cholesterol acyl transferasewith the proviso that the linked isolated peptides or mimetics thereofdo not consist of: (SEQ ID NO: 18)GFFSFIGEAFQGAGDMWRAYTDMKEAGWKDGDKYFHARGNYDAAQRGPGGVWAAEKISDARESFQEFFGRGHEDTMADQEANRHGRSGKDPNYYRPPGLP AKY; (SEQ ID NO: 19)GFFSFVHEAFQGAGDMWRAYTDMKEANWKNSDKYFHARGNYDAAQRGPGGVWAAEKISDGREAFQEFFGRGHEDTIADQEANRHGRSGKDPNYYRPPGLP DKY; (SEQ ID NO: 20)RSFFSFLGEAFDGARDMWRAYSDMREANYIGSDKYFHARGNYDAAKRGPGGVWAAEAISDARENIQRFFGHGAEDSLADQAANEWGRSGKDPNHFRPAGL PEKY; or(SEQ ID NO: 21) RSFFSFLGEAFDGARDMWRAYSDMREANYIGSDKYFHARGNYDAAKRGPGGAWAAEVISNARENIQRLTGHGAEDSLADQAANKWGRSGRDPNHFRPAGL PEKY.


81. The pharmaceutical composition of claim 79 wherein the isolatedpeptide or mimetic thereof which enhances cholesterol ester hydrolasecomprises a formula: (SEQ ID NO:  29)X₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁X₁₂X₁₃X₁₄X₁₅X₁₆X₁₇X₁₈

or a portion thereof wherein X₁ and X₉, X₁₂ or X₁₈ are amino acidscapable of forming a salt bridge; X₆ is glutamic acid or lysine or anamino acid which is a conservative substitution thereof; and X₂, X₃, X₄,X₅, X₇, X₈, X₁₀, X₁₁, X₁₃, X₁₄, X₁₅, X₁₆, and X₁₇ are independently anyamino acid.
 82. The pharmaceutical composition of claim 81 wherein X₂ isglutamine or an amino acid which is a conservative substitution thereof;X₃ and X₄ are independently alanine or an amino acid which is aconservative substitution thereof; X₅ and X₁₅ are independentlyasparagine or an amino acid which is a conservative substitutionthereof; X₇ is tryptophan or an amino acid which is a conservativesubstitution thereof; X₈ and X₁₁ are independently glycine or an aminoacid which is a conservative substitution thereof; X₁₀ is serine or anamino acid which is a conservative substitution thereof; X₁₃ is asparticacid or an amino acid which is a conservative substitution thereof; X₁₄is proline or an amino acid which is a conservative substitutionthereof; X₁₆ is histidine or an amino acid which is a conservativesubstitution thereof; and/or X₁₇ is phenylalanine or an amino acid whichis a conservative substitution thereof.
 83. The pharmaceuticalcomposition of claim 79 wherein the isolated peptide or mimetic thereofwhich enhances cholesterol ester hydrolase comprises: (SEQ ID NO: 4)DTIADQEANRHGRSGKDPNYYRPPGLPDKY; (SEQ ID NO: 8)ADQEANRHGRSGKDPNYYRPPGLPDKY; (D-form;SEQ ID NO: 10)ADQEANRHGRSGKDPNYYRPPGLPAKY; (SEQ ID NO: 25) ADQEANRHGRSGKDPNYYR;(SEQ ID NO: 11) ADQAANKWGRSGRDPNHFR; (SEQ ID NO: 12)ADQAANEWGRSGKDPNHFR; or (SEQ ID NO: 26) DQAANKWGRSGRDPNHFR,

or a peptide variant of one of these peptides or a portion thereof.84-85. (canceled)
 86. The pharmaceutical composition of claim 79 whereinthe isolated peptide or mimetic thereof which enhances cholesterol esterhydrolase activity and the isolated peptide or mimetic thereof whichinhibits acyl CoA:cholesterol acyl transferase are formulated togetherin a lipid complex or each formulated separately in a lipid complex andmixed together prior to administration.
 87. The pharmaceuticalcomposition of claim 79 wherein the isolated peptide or mimetic thereofwhich enhances cholesterol ester hydrolase activity and/or the isolatedpeptide or mimetic thereof which inhibits acyl CoA:cholesterol acyltransferase is linked to Z and wherein Z is a targeting agent, a secondagent for treatment of atherosclerosis, cardiovascular disease, orcoronary heart disease, or an agent which enhances solubility,absorption, distribution, half-life, bioavailability, stability,activity and/or efficacy of the compound.
 88. A pharmaceuticalcomposition comprising the compound Y—Z of claim 13 and and apharmaceutically acceptable vehicle. 89-105. (canceled)
 106. An isolatedpeptide comprising RGFFSFIGEAFQGAGDMWRAY (SEQ ID NO:7).